 SerialEM Help Index

General Topics ...

Introduction to SerialEM

Mouse and Keyboard Controls

Control Panels

Image Acquisition

Acquiring Tilt Series

Montaging

Low Dose Mode

Energy Filtering

Using the Navigator

Settings Files

Macros

Macro Commands

Setting Up SerialEM

Property File Entries

Commands

File menu

Settings menu

Camera menu

Calibration menu

Focus menu

Macro menu

Tasks menu

Tilt Series menu

Process menu

Navigator menu

Window menu

Help menu

Control Panels

Buffer Status panel

Buffer Control panel

Image Display Control panel

Microscope Status panel

Tilt Control panel

Camera & Macro Control panel

Image Alignment & Focus panel

Low Dose Control panel

Montage Control panel

Filter Control panel

Dialog Boxes

File Properties dialog

Camera Setup dialog

Gain Reference dialog

Montage Setup dialog

Tilt Series Setup dialog

Tilt Series Resume dialog

Tilt Series Back Up dialog

Navigator window

Macro Control dialog

Macro Editing window


Introduction to SerialEM

SerialEM presents an integrated environment for image acquisition, display, and storage, as well as for the control of the Tecnai microscope needed to acquire montaged images and tilt series.  You can acquire images from Gatan cameras using parameters that you set from within SerialEM.  Images can be zoomed, panned, and compared with each other using controls similar to those in Imod.  Successive images are conveniently saved to a single MRC file, ready for display in Imod.  The Tilt Series Controller will acquire a tilt series automatically using a prediction algorithm to minimize acquisition time and specimen exposure.  A low dose mode can be used to perform focusing and tracking away from the area of interest.  SerialEM can also automatically capture a montage of overlapping frames, and acquire a montaged tilt series.  The program also contains a macro feature for programming repetitive actions.

Controls: SerialEM is controlled through its menus, control panels, and hotkeys.  Menus contain commands related to a topic, as well as some entries to set parameters related to that topic.  The control panels provide status information, buttons for performing all frequently used operations, and buttons for setting parameters related to the particular panel.  Here is some general information about Control Panels.  Hotkeys are summarized in the section on Mouse and Keyboard Controls.

Buffers and display windows: Images are initially displayed in a main image display window.  Images are kept in a set of buffers, and the main display window can show an image from any one of these buffers.  Similarly to Imod, you can riffle through the images with PageUp and PageDn.  The buffers are referred to by letter.  Images from the camera are always placed in buffer A, the first buffer.  The program is typically set up to roll images through the first three buffers.  In other words, when a new image comes in, the existing image in buffer A is moved to buffer B, the image previously in buffer B is moved to buffer C, and the image in buffer C is lost.

It is also possible to copy an image to a new, free-standing window, which could be useful if you want to look at two images side by side.

Camera control: The camera controls are patterned after those available in Digital Micrograph, but there are some important enhancements that provide more flexible control of the specimen exposure, make it easier to select subareas of the camera, and allow control of whether a dark reference is taken.  General information about image capture is available in the section on Image Acquisition.

File storage: When images are saved to a file, successive images are typically stacked into a single file in the MRC format.  The pixel size is set in the header, and tilt angle and other information can be stored in the extended header area.  The file header is maintained after every image is stored, so that the file will be readable if something goes wrong.

Image alignment: Alignment of images is a key component of data collection.  SerialEM provides a linkage between the shifting of an image in a window, which is referred to as an alignment shift, and the physical realization of this shift using the image shift feature of the microscope.  This means that if you want to center a particular feature in the camera frame, you can impose an alignment shift on an image that has already been acquired, simply by dragging it with the right mouse button.  The microscope image shift is then changed by the right amount so that the next image that you acquire appears in the desired place.  Two images can also be aligned to each other by cross-correlation.  This is referred to as autoaligning.  The image in buffer A is correlated to a reference image in another buffer.  Before doing this correlation, the program will stretch the image taken at a higher tilt angle to give a better match to the other image.

Autofocusing: SerialEM determines the defocus of the specimen by the standard technique of measuring how much the image moves when the beam is tilted.  When the specimen is in the focal plane, its image does not move; and the farther it is from focus, the bigger the beam-tilt induced movement.  For this to work correctly, you need to align the beam tilt pivot points properly, a standard step in the Direct Alignments used to tune the microscope.  Be sure that the specimen is at the eucentric height and at minimum contrast focus when you tune the pivot points.  They do not need to be perfect, but do need to be close.  Measurement of defocus is typically done by taking a picture with positive beam tilt, one with negative beam tilt, then another with positive beam tilt.  Having three pictures allows the program to compensate for drift.  Once the program has measured the defocus, it can change it to achieve the level of defocus that you specify.  This procedure is called autofocusing.


Mouse and Keyboard Controls

Mouse actions in the image display window:

Left mouse button click: place a marker point in the image and print the coordinates and value in the middle panel of the status bar.  Several procedures make use of the position of a marker point.

Ctrl – left mouse button click: zoom up.

Ctrl – right mouse button click: zoom down.

Left mouse button drag: Holding the left button down and moving the mouse will pan the image, if it is zoomed bigger than will fit in the window.

Right mouse button drag: Holding the right button down and moving the mouse will change the alignment shift of the image.  If the image is in buffer A, this will also change microscope image shift so that the next image acquired will match the image with this alignment shift.  If there is a marker point on the image being aligned to, its position will show up in red.  There is an option available to have particularly large moves with the right mouse button move the stage instead of change the microscope image shift.

Shift - right mouse button drag: Shifting the image in buffer A while holding the Shift key down will result in the stage being moved, rather than microscope image shift being changed.

Hotkeys:

-/_	Zoom an image down

=/+	Zoom an image up.

R	Resize the main image window to fill the SerialEM frame.

Ctrl A	Autoalign

Ctrl D	Tilt Down

Ctrl F	Acquire image with Focus parameters

Ctrl G	Autofocus

Ctrl H	Halt camera acquisition

Ctrl I	Image information – min/max/mean and SD.

Ctrl L	Acquire image with Preview parameters

Ctrl M	Start a montage

Ctrl O	Open old image file

Ctrl P	Open camera parameter dialog box

Ctrl R	Acquire image with Record parameters

Ctrl S	Save image to file

Ctrl T	Acquire image with Trial parameters

Ctrl U	Tilt Up

Ctrl V	Acquire image with View parameters

Space bar	Stop or restart continuous image acquisition.

Esc	Stop camera acquisition and any running tasks.

F1	Open context-sensitive help when the mouse is over a menu item or a dialog box.

Shift-F1	Will allow you to click on an item and open help for it.

Ctrl F1	Run macro 1

Ctrl Fn	Run macro n, n = 1 to 10

The keys in the 6-key cluster above the arrow keys do the following:

PageUp	display the previous occupied buffer (a lower letter).

PageDn	display the next occupied buffer (a higher letter).

Home	display buffer A, or the first occupied buffer.

End	display the last occupied buffer (highest letter).

Insert	display the buffer that is being autoaligned to.

Delete	display the buffer that is being read into from file.

The four arrow keys will pan the image by small steps if it is zoomed bigger than the window.


Image Acquisition

An image is captured from the CCD camera with a sequence of 3 steps: clearing the CCD chip, exposing the camera to the beam and integrating charges for some period of time, then reading the image off of the chip.  The readout is done line by line, so it is often very time-consuming.  The clearing time is also substantial because the chip is cleared by shifting accumulated charges to one edge.  Clearing times are about 0.7 to 0.9 second for 2K Megascan or 4K single-port readout Ultrascan cameras, 0.25 second for a 4-port readout Ultrascan, or very short for some current 1K cameras.  It is important not to have a beam on the camera during the clearing time, because then charges will build up as they are being removed, adding a ramp of intensity to the image that is subsequently acquired.

Many plastic-embedded specimens show a transient shift of the image when the beam is turned on to take an exposure; this drift produces a smeared image.  To take good images, you need to use drift settling, which provides an initial exposure of the specimen to the beam just before the image is acquired.  This initial shift is worse on slot grids than on mesh grids and tends to be worse at high tilts.  It seems to depend on the total beam hitting the sample rather than the brightness of the beam in the area of interest.  This means you should work with the largest spot size that gives you enough beam.

Exposing the specimen to the beam before the exposure without exposing the camera during its clearing time requires the use of at least two shutters, one below the specimen and one above.  The traces below show how DigitalMicrograph manages the two shutters in standard shuttering mode with the alternate (beam) shutter normally closed.  A trace goes high to indicate when a shutter is open or beam is on the camera.  Although this shuttering provides some pre-exposure, there are two problems here.  First, the amount of pre-exposure is fixed at the clear time, which could be too short or longer than needed. Second, the beam is left on the specimen during camera readout.  (Note that this occurs only when using the shuttering mode that provides pre-exposure and does not happen if you are just using the beam shutter for low-dose exposures.)

DigitalMicrograph does provide a way to increase the drift settling, but this has problems of its own.  As shown in the following traces, the extra settling is implemented by keeping the beam on the CCD during the clear time.  This introduces an intensity ramp in the image, which is eliminated by having the beam on for the equivalent time during the “dark” reference.  These ramps use up some of the dynamic range of the camera, but worse than that, the dark reference contains image features and will become invalid when exposure or specimen location changes.

To deal with these problems, SerialEM has implemented a shuttering method using the beam-blanker available through the Tecnai interface.  As indicated in the two sets of traces below, this extra shuttering can provide a flexible range of drift settling times and also eliminates the many seconds of exposure during camera readout.

Low-noise CCD cameras are inherently slow, so you should be aware of what governs image capture time and how to speed it up.  The total capture time is the sum of: any drift settling time above the clearing time, the clearing time, the actual exposure time, time to read out the image, and time to process and display it.  Drift settling of less than the clearing time is subsumed in the clearing time.  Processing time is minimal, so you should always use gain normalization to remove intensity variations due to imperfections in the phosphor screen.  Read-out time is proportional to the number of pixels of data being read out of the CCD chip.  It is about 12 seconds for the 2K x 2K pixels through a single readout port, so binning, which is done on the chip before read-out, can speed up the read-out tremendously.  The read-out time is actually dominated by the number of lines being read, so for a given number of pixels being read, the read-out is fastest if those pixels are in the fewest number of lines.  This is the reason for the Wide Quarter and Wide Half areas provided by the buttons in the Camera Setup dialog.  Their readouts are only slightly longer than the readout for square images that are half as big.


Acquiring Tilt Series

The basic steps involved in acquiring a tilt series are quite simple: tilt by an increment, adjust image position to keep features of interest centered, adjust focus to maintain a desired level of defocus, and acquire and save an image.  However, several factors make this process considerably more complicated.  First, it is highly desirable to skip some of these steps as often as possible, because of the time and specimen dose required to perform them on every tilt, yet it is not trivial to skip steps without jeopardizing the quality of the tilt series.  Second, the need to track image position with a lower magnification image when working at a high magnification, or with an image away from the area of interest when trying to minimize dose, makes this operation more complex.  Third, things do go wrong that require manual intervention, and the program must have the flexibility to allow for a variety of interventions.

The part of SerialEM that manages tilt series acquisition is called the Tilt Series Controller (TSC).  It deserves to be thought of as an entity in its own right because it takes control of many aspects of program operation.  For example, you will not be able to tilt, copy images to certain buffers, or save images directly while the TSC is in control – you can do these things only through the TSC.

When the TSC is running, buffer usage is standardized as follows: images roll from buffer A through buffer C as new images are acquired; buffer D is the primary buffer for autoalignment, buffer E is a secondary buffer for aligning low magnification images or Trial images in low dose mode; and buffer F can be read into from a file.  Copying to buffers E and F is disabled.

You can suspend a tilt series to change the image alignment or focus.  See the help for the Tilt Series Resume dialog box for more details on what options are available when you do this.  You can also back up to previous tilt angles; see the help for the Tilt Series Back Up dialog box for more details on this process.

Minimizing tracking errors

Running a tilt series continuously from one extreme tilt to the other can result in the area of interest not being well centered at zero tilt.  This can happen either because of inaccurate tracking from high to low tilt, or because it was difficult to recognize the correct center position at the starting tilt angle.  SerialEM has several features that can help prevent this problem.  One is the ‘Walk up’ procedure, which allows one to start at zero tilt (or a higher tilt if desired) and tilt up to the starting angle in a series of coarse increments with tracking images that are successively aligned to each other.  This could be particularly useful when it is hard to recognize the features of interest at high tilt.  You can activate the ‘Walk Up’ procedure yourself (from the Tasks menu) before you start the TSC, or you can activate the TSC at a low tilt angle and have it walk up to the starting tilt angle.

The second feature that may help tracking is called an ‘anchor’.  This is an image taken at around 40 to 50 degrees that the TSC can use as a reference when the tilt series reaches that angle.  The angle should be low enough so that tracking is likely to be accurate from that point downward, but high enough so that uncorrected Y-axis errors above that point are unlikely to cause data loss in the reconstruction.  (Note that because the region of interest is foreshortened in the Y direction at high tilt, alignment errors can be quite high in Y without losing any of the region of interest.)  Angles in the range of 40-50 degrees should satisfy these criteria.  If you are starting the TSC at a low tilt angle and having it walk up to the starting angle, you can have it leave an anchor at an appropriate angle.  If you are going to high tilt before starting the TSC, you can also specify an angle to leave an anchor at when you use the ‘Walkup & Anchor’ procedure.

The fallback feature for fixing a tracking error is manual intervention.  Simply stop the series with the ‘End’ button.  It will stop with a Record image in Buffer A.  Shift this image to the desired alignment with the right mouse button.  Then select the ‘Resume’ option, check the box ‘Use image in buffer A as reference for alignments’ in the Tilt Series Resume dialog box, and push the ‘GO’ button.

Starting a tilt series

The TSC can be started either at a low tilt angle or at the starting tilt angle.  You can safely start at low tilt if you are fairly confident that:

•	The camera parameters (exposure time and drift settling) are suitable for getting good pictures at high tilt.

•	Autofocusing will work well enough to find focus at high tilt.

•	The beam can be adjusted automatically to give the desired counts in the image without becoming too small and encroaching on the camera area.

If you have doubts about any of these factors, you should go to high tilt before starting the TSC, and make sure that images look good, that the specimen is focused, and that the beam can be adjusted properly.  These conditions may be satisfied if you have already done one tilt series on the specimen, in which case letting the TSC start from low tilt would save some effort.

Here is a sequence of steps for starting a tilt series:

1.	Decide if you are going to start at low tilt or go to high tilt before starting the TSC.

2.	If you are going to high tilt before starting the TSC, refine eucentricity.  Select the ‘Refine and Realign’ command if you have already centered your region of interest and plan to use the ‘Walk Up’ procedure; otherwise ‘Fine Eucentricity’ is sufficient unless the magnification is high enough that you might lose your selected area.

3.	Go to high tilt.  Consider using ‘Walkup & Anchor’ to save an anchor, as described above.

4.	Make sure that the exposure settings for Focus, Trial, and Record give images without drift.

5.	Do autofocus and see if it gives a well-focused Record image with the best focus in the center of the field. If not, you can use the Move Focus Center command in the Focus menu to move the center of focus to the middle of the field, or you can vary focus by hand.  Once focus is found, you should run the Measure Defocus command in the Focus menu and use the reported value to set the target defocus.

6.	Set the beam intensity to give the desired level of counts.  You can do this by hand or use the Set Intensity command in the Tasks menu.  This can be done with either a Trial or a Record image in buffer A.  When you select the command, the message in the dialog box will show the mean counts that a Record image would give with the present beam intensity.  You can enter either a new number of counts, or a factor by which to change the intensity.  If you change the number by much (more than 10-20%), you should take another image and look at the beam on the screen to make sure it is not too small.  If you cannot comfortably achieve the desired value, just adjust the beam so it is large enough.  You will be able to select an option to reach the desired target number of counts at a lower tilt angle, and the program will reach this target as fast as it can without making the beam smaller than this initial setting.

7.	Take a final Trial image and center the features of interest.

8.	Open the Tilt Series Setup dialog box in the Tilt Series menu and check the entries from top to bottom, paying particular attention to the following:

9.	Check the starting and ending angles and angular increment.

10.	Make sure the delay time after tilting is appropriate for the holder and tilt increment (small increments need less delay time; heavy holders need more).

11.	Decide whether you need low magnification tracking.  This is recommended for magnifications above 50,000, and might be needed at a somewhat lower magnification if you are starting at a particularly high tilt angle (say, above 70 degrees).

12.	Make sure the limit on image shift is appropriate for the objective aperture size and other conditions.  On the 300 KV Tecnai, operating at a relatively low magnification, montaging, and using low dose may all require a smaller limit on image shift, perhaps as low as 1 micron.  When the limit is reached, the TSC will automatically reset the image shifts, move the stage to compensate, and recover from the physical disturbance caused by this movement.

13.	Make entries for beam intensity control.  Checking ‘Do not increase intensity above value for first saved image’ is almost always a good idea.  If you are starting at high tilt and needed to set the beam to give less than the desired target number of counts, check ‘Keep intensity below current value (use if intensity already set up)’.  This will achieve that target as soon as possible without overconstricting the beam.  On the other hand, if you are starting at zero tilt and know that you need to reduce the number of counts at high tilt, fill in the desired target number in the ‘Set intensity to keep mean counts of Record images at’ text box, check the ‘Taper counts down to’ option, and put your reduced target for the highest tilt in the text box there.

14.	If you have had difficulty with autofocus at high tilt, you can select the ‘Focus every time above’ option and fill in the desired angle above which you want to focus on every tilt.

15.	If you have a centered image in buffer A, select the option to ‘Align to image now in Buffer A’.

16.	If you are starting at zero tilt, you should select the option to ‘Refine eucentricity first’ unless you have already done this.

17.	If you are starting at high tilt and have acquired an anchor image on the way up, select the ‘Use mid-tilt anchor’ option and then use the up or down arrow if necessary to indicate which buffer contains the anchor (it should be in the right place if you used ‘Walkup & Anchor’).

18.	Under ‘Tracking control parameters’, you typically need to consider only the first line, which controls whether Record images will be repeated if they are not centered well enough.  If you never want this to happen, unselect the option.  The percentage here will determine the width of the area near the edge of your reconstruction that is degraded because some images fail to contribute data to it.  A value of 5% is probably reasonable for most uses.  If the structures of interest nearly fill the camera frame at low tilt, then you might want to reduce this to 2.5-3%.  If you are working at very high magnification, you might want to raise the value or unselect the option.

19.	If you are working at high magnification where tracking will be relatively less precise, you may need to increase the value for ‘Get tracking image when error in X/Y prediction is >’ to avoid excessive tracking images.  You may also need to increase the criterion for ‘Get new track reference if Record alignment differs’.

After you start the tilt series, the TSC will perform a number of preliminary actions, which may include finding eucentricity, walking up, finding focus, getting a first reference image, and adjusting intensity.  Also, the program will use the Reverse Tilt procedure to eliminate tilt backlash, so there is no need for you to do this yourself.  This procedure involves tilting up and back by about 3 degrees, with lower magnification pictures taken before and after to track the potentially large shift in position that can occur.  If there is ‘pole touch’ in this process, the program will try to compensate for this by taking lower magnification tracking images during the first few tilts, until the backlash is worked out.

How predictions work

The TSC minimizes the number of tracking and focusing positions by predicting where the specimen will be after each tilt.  It predicts position separately in the X and Y directions (along and across the tilt axis) and uses image shift to compensate; it predicts the position in Z and changes focus to compensate.  At the beginning of the tilt series, the information needed to make reliable predictions accumulates gradually.  After two tilts, there is enough information to make simple extrapolations, but not to know how accurate they are.  With data from three or more tilts, the program can assess the accuracy of a prediction from how well the positions fit a straight line.  In addition, if predictions were made on the previous tilt, the program can determine how far off they were.  Thus, at a given tilt angle, there are two kinds of prediction errors available: the computed error of the prediction for the next angle, and the actual error in the prediction for the current angle.  Only when both of these errors are low enough will the TSC rely on a prediction and skip a tracking or focusing step.

The accuracy of the prediction generally increases as more data become available, but only as long as the positions change regularly.  Because the specimen and stage do not behave ideally, it is necessary to restrict the positions used for the predictions to those from the most recent tilts.  For each axis, there is a maximum tilt range over which positions will be fit (a parameter that can be set in the Tilt Series Setup dialog box.)  Moreover, the TSC may restrict the tilt range used for a particular fit even more if it substantially in increases the accuracy of the prediction.  The number of points dropped from a prediction fit is reported as ‘nDrop’ in the line describing the prediction.  These limitations are expressed in terms of tilt range rather than number of tilts because the deviations from ideal behavior that limit the quality of predictions probably occur over a certain range of motion independent of the number of steps.  If you have smaller a tilt increment, the predictions should be good over a larger number of tilts.

Certain events are considered to disturb the predictability of the system and will cause the TSC to ignore some or all of the position data that are available.  These events include resetting the image shift, tilting back to redo a Record image, and changing the alignment reference.  If you stop then resume a tilt series, the TSC will try to determine whether you have done something that would jeopardize the predictions.


About Montaging

Montaging allows one to capture an array of multiple, overlapping frames automatically using either electronic image shift or stage movement.  Each such frame is called a piece.  Pieces are numbered and referred to by their position in X and Y within the montaged image (numbered from 1) and by their section number or Z value (numbered from 0).  For example, in the first set of images making up a 2 by 3 montage, piece 2, 2 is the middle piece in the right column, and the Z value would be 0.  All pieces in a montaged image have the same Z value.

Montaging can be initiated in several ways: by selecting Montage Parameters from the File menu, by selecting Montage or Prescan from the Camera menu, or by pressing the Start or Prescan buttons in the Montage Control panel .  Whichever way is used, you will then encounter a series of dialog boxes: the Montage Setup dialog box for defining the montage size, File Properties dialog for specifying how images are stored, and the Save As dialog box to specify the output file.  Montaging can also be restarted on an existing file simply by reopening that file.  In this case, you will enter the Montage Setup dialog box to see the parameters governing the montage, but you will not be able to change most parameters.

You should always calibrate the image shift at the given magnification before starting to acquire montages for a tilt series.  See the Image Shift command in the Calibrate menu.

When montaging is started, the Montage Control panel opens up.  At that point, a montage can be acquired by pressing the Start button there or the Montage button in the Camera & Macro Control panel, or by using the Ctrl M hotkey or the entry in the Camera menu.  Images are always acquired with the ‘Record’ camera parameters.

The Prescan option, available in control panels and the Camera menu, will acquire binned down images at all piece positions and use them to compose an overview image.  These images are acquired relatively quickly and are not saved to file, so this is a good way to find out what features are included in a montaged area without taking actual montages.

To acquire a montage, the program shifts to each of the frames and acquires and saves an image.  As it goes, it composes an ‘overview’ image, a single, binned down image of the entire montaged area.  This image is left in buffer B.  The different pieces may not be shifted into perfect registration when composing the overview, so this image may show some sharp transitions between the pieces.  This should not be a cause for concern.  There is an option in the Montage Control panel to have pieces shifted into register instead.

You can use the right mouse button to impose an alignment shift on an overview image to reposition the field of view.

As it acquires frames, the program also uses cross-correlation to measure the misregistration between each pair of overlapping pieces.  When the whole montage has been acquired, it uses these errors in registration to determine how to shift all of the pieces so as to minimize the error.  The program informs you in the Log window of the error before and after shifting pieces into best registration.  The error before shifting can become high (more than 10 pixels) if there is drift during the acquisition or if image shift is not well calibrated.  The error after shifting should be low (under 0.5 pixel) at low tilt angles, but may become relatively high (1-4 pixels) at high tilt because image distortions prevent pieces from fitting together well.  A high error can also occur if there is an error in the correlation between some pair of pieces.  The latter problem can be corrected afterwards in Midas.

After acquiring a montage, the program also composes a ‘center’ image and leaves it in Buffer A.  For a montage with an odd number of pieces in X and Y, this would just be the center piece, but in other cases it composes this image from halves of two overlapping pieces or from quarters of four overlapping pieces.  The misregistrations between pieces are taken into account in fitting these pieces together, so the center image should not show sharp transitions and should be suitable for aligning from one tilt to the next.

You can reconstruct the overview and center pieces for a stored montage by simply selecting the Read command from the File menu.  The program will go through the same sequence of operations using each piece from the file that it does with newly acquired pieces, leaving a center image in buffer A and an overview in buffer B.

If the range of image shift required to make a montage is large, you can use stage movements instead.  This is specified by an option in the Montage Setup dialog box


About Low Dose Mode

The essence of low dose mode is that focusing and tracking operations for a tilt series are done in a location separate from the area being recorded, to spare that area from unnecessary beam exposure.  In order to provide accurate focusing and tracking, these separate areas must be displaced along the tilt axis from the center of the recording area.  SerialEM’s low dose mode provides four areas that can be defined to have different settings of magnification, spot size, and beam intensity, centered in up to 3 different locations, as follows:

1.	Record area – the area where the region being recorded is located.  The magnification and beam should be set up for the final image acquisition.  Both Record and Preview capture images from this area; the Record camera parameters would be set up for the final acquisition, while Preview parameters would be set up for a highly binned, low exposure image.

2.	View area – this area is centered on the Record area.  The magnification should be low for several reasons: to provide an overview of a larger area, to minimize specimen exposure, and to provide reliable tracking in the various SerialEM ‘tasks’ that require low magnification views.  Taking an image with the View camera parameters will acquire from this area.  View camera parameters should be set up for a binned, low exposure image.

3.	Focus area – this area is displaced along the tilt axis from the Record area.  The beam must be set up so as not to intrude on the Record area.  Taking an image with the Focus camera parameters will acquire from this area.

4.	Trial area – like the Focus are, this area is displaced along the tilt axis and the beam must be constricted.  Taking an image with the Trial camera parameters will acquire from this area.

Although the Focus and Trial areas can be set up with completely independent parameters, it is most convenient to constrain them to be identical.  This saves the effort of setting up the beam for both areas, avoids the image shift settling time required for getting between them, and minimizes the total range of image shift occupied needed for low dose work.  The latter factor is currently important on the 300 KV Tecnai because the objective aperture begins to occlude the image area for relatively small image shifts.

In low dose mode, numerous features of SerialEM operate differently.

•	Tasks that use low magnification tracking (finding eucentricity, reversing tilt, and resetting image shifts) will automatically use View images.  Walking up will use images of the Trial area.

•	Autoalignment operates with two autoalign buffers.  A Record or Preview image will be aligned to the image in the first autoalign buffer, whereas a Trial or Focus image will be aligned to the second autoalign buffer.  The ‘Align to’ button in the Image Alignment & Focus control panel will indicate which are the two autoalign buffers.

•	Autoalignment will work with the beam constricted inside the camera field because autoalign will detect dark areas in the corners of the image (namely, areas outside the beam) and use only the largest rectangle inside these areas for alignment.

•	The Tilt Series Controller uses D and E as the two autoalign buffers.  It uses images of the Trial area for tracking, but it also maintains a reference image from the Record area, so that each new Record image is aligned to that reference and then replaces it.

•	If you choose to adjust intensities automatically during a tilt series, only the intensity of the Record area will be adjusted.

You can set the properties of a low dose area by making it be the current area then adjusting its features.  The current area and its beam properties are displayed on the second line of the Low Dose control panel .  There are two ways to set the current area: take an image of the area, or put the screen down and select the area with the ‘Area to show when screen down’ radio buttons.  You can adjust the properties of the current area by turning on the ‘Continuous update of mag & beam’ checkbox.  With this option on, any changes in the magnification, spot size, condenser lens setting, or energy filter settings will change the defined properties of the area.  If the option is off, you can change these features temporarily, but the stored properties of the area will be reimposed next time a picture is taken of the area.

If you want to make initial adjustments to the properties of an area with no exposure of the specimen at all, you can turn on the ‘BLANK BEAM while screen down’ checkbox, then lower the screen, select the area with a radio button, and adjust its features.

The positions of the Focus and Trial areas can be set by selecting the Focus or Trial radio buttons in the ‘Define position of area’ box.  Once one of these buttons is selected, two pieces of information become available at the bottom of the Low Dose control panel .  One is a text box showing the distance along the tilt axis from the Record area to the area being defined, where negative and positive numbers reflect opposite directions along the axis.  The other display shows essentially a safety factor for spillover of the beam from the area being defined into the Record area; it is the distance between a circle circumscribing the area being defined and the edge of the Record area.

There are several ways to adjust the position of the area being defined.  In addition to simply typing in a distance in the text box, the most convenient is to take a picture of the View area.  The position of the area being defined relative to the center of the View will be shown by a green cross, and you can click with the left mouse button to select a new position.

The final concept that needs explaining is that of balancing the shifts.  On the 300 KV Tecnai, one should think of the objective aperture as defining a circle within which the specimen can be imaged by means of image shift.  If the Record area is in the middle of this circle, then the Focus and Trial areas will be displaced to near the edge of the circle, and there may not be much range of image shift left for tracking during the tilt series.  Given these limitations, it is preferable to arrange the Record and Trial areas so that the center of the circle is midway between them, because then neither one will be as close to the edge of usable circle as the Trial area is with the Record area in the center.  This is accomplished by pressing the ‘Balance Shifts’ button.  The change can be undone by pressing the ‘Center Unshifted’ button, so-called because it will recenter the areas that are considered unshifted (Record and View).

Your low dose parameters are saved to your settings file and will be restored when you restart SerialEM.  If you have never started low dose before, the areas are undefined, and they will acquire the current microscope settings when they are first activated.

Steps for Initially Setting up Low Dose

It is somewhat difficult to get low dose set up initially, but following a set procedure may be helpful.

1)	Before going to low dose mode, set the magnification and beam strength that you will want for the View area.

2)	Turn on low dose mode and take a picture of the View area.

3)	Turn on ‘Continuous update’ to adjust the View area properties further.  Be sure to set any View-area-specific GIF settings.

4)	Copy View properties to the Record area with the ‘R’ button in ‘Copy current area mag & beam’ radio group.

5)	Take a Preview or Record picture to go to the Record area, then turn on ‘Continuous update’.

6)	Adjust the magnification for the Record area, and any specific filter settings.

7)	Adjust the beam strength by taking a Preview or Record and selecting ‘Set intensity’ in the Tasks menu.  Enter the desired number of counts in a Record image.  If there is a big change, this may take 2-3 iterations.

8)	If the beam is too small, reduce the spot size; if it is too large (i.e., you are concerned about extra exposure to the Trial/Focus areas), then increase the spot size.

9)	If you want to get the beam to be not much bigger than the camera field of view, you need to condense it so that you can see the whole beam, then expand by a controlled amount.  See step 14.

10)	Make sure ‘Keep Focus and Trial identical’ is selected and copy Record properties to the Trial area with the ‘T’ copy button.

11)	Select the radio button to define the position of the Trial area.

12)	Take a View picture.  Click with the left mouse button to define a position for the Trial area.  Note the ‘Maximum area separation’ and click again to get a sufficiently positive separation, or just type in a position.

13)	Take a Trial picture.  Adjust area-specific filter settings (e.g., slit out).

14)	Here it is essential to get the beam constricted to just the Trial area and centered on it.  You can do this either by adjusting brightness and position blindly or by using the ‘Set Intensity’ and ‘Move Beam’ operations in the Tasks menu. When using ‘Set Intensity’, note that changing intensity by a certain factor should change beam diameter by the square root of that factor.  Also note that your changes will not be accurate when the beam does not fill the camera field.  In general, the procedure is:

a.	Constrict the beam enough so that you can see enough of its edge to be able to center it.

b.	Change spot size if necessary to get close to the desired intensity.

c.	Center the beam.

d.	Expand the beam until it just circumscribes the area.

If you are restarting with some existing parameters, you would not do all of these steps.  You would probably take a View and adjust its properties if necessary (steps 2 and 3), take a Record and adjust its properties (steps 5-9), then adjust the Trial/Focus area (steps 11-14).


Energy Filtering

SerialEM allows convenient use of the energy filter (GIF) and efficient tilt series acquisition with the GIF.  It relies on the EFTEM software being installed in the Tecnai, but provides a number of additional features:

•	Automatic switching between regular lens mode and EFTEM lens mode when the screen is raised or lowered.  For the occasions when you need to see the constricted beam of EFTEM lens mode on the screen, you just toggle this automatic switching off.

•	Automatic switching between upper camera and GIF camera.

•	Automatic changing of magnification to provide the least change between the pixel sizes on the upper camera and on the GIF camera.

•	Automatic intensity adjustment so that the electrons per pixel per second will be the same on the GIF camera as on the upper camera.

•	Integration with low dose mode so that different filter settings can be used for pictures of the different low dose areas.

Even without SerialEM, there are numerous ways to control filter settings: the EFTEM panel in the Tecnai User Interface (TUI), the Autofilter palette in DigitalMicrograph, and the Filter Control program itself.  SerialEM tries to be a good citizen among this crowd and respond to changes made in other interfaces.  FEI recommends that the filter be controlled only through TUI and hides the Autofilter palette to enforce this.  This is probably sound advice for any actions that cannot be done through SerialEM.  Specifically, this includes aligning the zero loss peak (ZLP), tuning the GIF, and performing spectroscopy.

It is important to distinguish between SerialEM’s EFTEM mode and the Tecnai EFTEM lens mode.  When EFTEM mode is on in SerialEM, there are many implications, but the Tecnai may not actually be in EFTEM lens mode unless the screen is raised.

The biggest problem in using the GIF for tilt series is that there may be an energy shift when changing to lower magnifications for various alignment tasks.  If the slit is in, the zero loss peak may shift out of the energy range of the slit, and the task will fail.  One solution would be to remove the slit when going to lower magnification, but this would cause an unpredictable and potentially large increase in intensity that could saturate the camera.  The solution in SerialEM is to calibrate the energy shifts and apply these automatically when changing magnifications, so that the slit can be left in and will still be centered at the lower magnification.  The program will also apply energy offsets to accommodate changes in slit width.  These capabilities allow you to explore different magnifications and slit widths without having to align the ZLP repeatedly.

SerialEM implements the three standard imaging modes: unfiltered (with the slit out), filtered to record only zero-loss electrons, and filtered to record electrons with a specific range of energy losses.  Once filtering is selected, it is possible to switch between zero-loss and a specific energy loss simply by toggling the ‘Zero loss’ checkbox; however, if you want a different slit width in the two cases you need to adjust it each time you switch between them.  See Filter Control panel for more details.

The Filter Control Panel in SerialEM includes a button to refine the alignment of the ZLP.  The procedure is referred to as a refinement because it will work only if the ZLP is already close to aligned, say within 10 eV.  The Align ZLP function available from TUI must be used for initial alignment, because it is capable of finding the ZLP from many eV away.  However, the refine ZLP procedure will align the ZLP even when it is a very small fraction of the total electrons, because it concentrates on the drop in signal as the slit is moved off of the left edge of the ZLP.  Thus, once a thick specimen is in, the refine ZLP procedure may be more reliable than the Align ZLP procedure run from TUI.

Steps for Using the GIF

1)	Be sure to start Filter Control, then DigitalMicrograph, then SerialEM after DigitalMicrograph reports Dynamic cameras registered in its Results window.

2)	Go to a mid-range magnification like 20,500 with the screen down.  Adjust the beam to a spot size and screen current that will get you through the tuning procedure without having to adjust intensity.  (Specific values vary widely; if you have to turn the beam down in the First Order isochromaticity adjustment, put the screen down when you get through the tuning and note the current for the next time.)

3)	Activate EFTEM mode in the Filter Control panel  and take a picture through the GIF just to verify function.

4)	Set the slit width to 10 to 20 ev; 20 seems preferable.

5)	Press ‘Align ZLP’ on the Tecnai EFTEM panel.

6)	Press ‘Tune GIF’ on the Tecnai EFTEM panel.

7)	Switch to the magnification that you will be using for final image acquisition.  Lower the screen and adjust the beam to fill the screen with enough intensity for a good gain reference (5 - 10 na?).

8)	Raise the screen and take a gain reference.

9)	Now you should assess the adequacy of the calibration of mag-dependent energy shifts (unless you will be working in low dose mode and having the slit out for pictures of the View area).  To do this:

a.	Set Trial parameters for a fast picture with moderately high counts, e.g. 256 x 256, center half area, 0.05 – 0.1 second exposre.

b.	Turn on intensity zoom.

c.	Set slit to 15 ev.

d.	Go to a low mag like 5600.  Take pictures with the slit in and the slit out and measure the mean counts (Ctrl-I).

e.	Repeat for other low mags that may be used in tasks (e.g. 4500 and 3000).

f.	If you see some serious beam loss or dark spots on the edges with the slit in, you need to calibrate the mag-dependent shifts, as described in  Mag Energy Shifts .  Enter the highest mag that you will use and accept the defaults for the other parameters.


Using the Navigator

The Navigator window and its associated menu entries provide the capability to save stage positions and return to them, to make and save maps of areas of interest, mark positions on these maps and go to the marked positions, and transform positions after moving the grid in the specimen holder.  This section explains some of the concepts involved in using the Navigator.

The Navigator maintains a list of items which are shown in a box.  It considers items to be of three types: points, polygons, and maps.  A point is a single location, which will be displayed with a colored cross on images that contain the point.  A polygon is set of connected points enclosing a region, which will be displayed as a set of lines forming a closed contour.  A map is an image that has been saved to a file; the Navigator keeps track of where the image was saved as well as the stage position of the image.  The location of a map is displayed as a rectangle on other images.  The Navigator makes it easy to reload a selected map image, regardless of whether all of your maps are in one file or separate files.  Maps can be either single frame or montaged images.

Points can be added in two different ways. You can move the stage to a specific location and then press a button (‘Add Stage Pos’) to add the location as a point.  Alternatively, once you have taken a low-mag image at a known stage location, you can press a button (‘Add Points’) to start adding points, then click the left mouse button at a series of locations in the image.  Each location becomes a new point item with a separate stage position deduced from its location in the image.  A polygon can be generated in a similar fashion, by pressing ‘Add Polygon’ and clicking points on an image.

A map can be created simply by pushing a button after acquiring an image and saving it to a file.  A montaged image is already saved to a file and can be made into a map immediately after being acquired, as long as the montage overview is still in buffer B.  There is also a feature for automatically acquiring a series of maps at selected points.  Again, these maps can be either single frame images or montages.  They will all be saved into a single file that must be set up before starting the automatic acquisition.

The large-area images used for maps are usually generated by taking montages with stage movements between the frames.  The Navigator menu provides three ways to define the area that will be acquired in a montage.  One option is simply to define an area that will capture the entire grid (‘Setup Full Montage’).  Another option is to move the stage to a series of points at the corners of the region of interest, such as the corners of the first and last sections of a ribbon.  When each of these points is marked as a ‘corner’ point, the ‘Setup Corner Montage’ menu entry will set up a montage to encompass all of the area bounded by these points.  The third option is to draw a polygon; the ‘Setup Polygon Montage’ menu entry will set up a montage to capture the area delineated by the polygon.  Note that in the latter two cases a full rectangular array of frames need not be acquired; the program will omit pieces that fall outside of the defined area.

The final concept that needs explaining is ‘registration’.  A registration is essentially one position of the grid in the specimen holder, or one relationship between positions on the grid and stage coordinates.  Every item in the Navigator’s list, as well as every image that you take, is associated with a registration.  When you first start using the Navigator, all of the items that you add to the list are assumed to be at the first registration.  If you disturb the grid in the holder (or even if you remove and replace the holder), you have changed the correspondence between positions on the grid and stage coordinates.  You should then indicate to Navigator that the registration has changed by increasing the current registration number.  At this point, items from previous registrations will no longer appear on newly acquired images (unless you select an option to make them appear) because they would not be in the right place.  To get them into the right place and make them useful again, you need to transform them to the new registration.

To transform items to a new registration, you must first define some of the Navigator points at the first registration as registration points.  You do this by selecting the ‘Registration point’ checkbox and, if necessary, specifying a point number as well for each of the relevant points.  (Registration point numbers will increment automatically.)  After you have gone to a new registration, you then move the stage to the same positions, add a new Navigator point at each, and identify each point as a registration point with the same number as in the first registration.  Once you have identified a set of corresponding points in this way, select a menu entry to transform the items from the first registration to the new one.  The program will find a transformation whose complexity depends on the number of registration points available.  Just a shift will be found with 1 point, a rotation and shift with 2 points, rotation, scaling, and shift with 3 points, or a full linear transformation including stretching with 4 points.  All items except the registration points will then be transformed and associated with the new registration.  (The registration points are left behind to allow the data to be transformed back if necessary.)  If you just need to adjust by a shift after removing and replacing the holder or after moving the stage around a lot, you can use just one registration point.

A suggested procedure for using the Navigator for mapping would be:

1.	Go to a magnification that can be used to map the area of interest in a reasonable number of large pixels, on the order of 10K x 10K (e.g., 10 by 10 pieces, each 1K x 1K).  This might be as low as 300x.

2.	Move the stage to the corners of the area of interest and add the stage position as a point at each corner.  Define each point as a corner point, then select ‘Setup Corner Montage’.  Alternatively, especially for cryomicroscopy, select ‘Setup Full Montage’.

3.	Acquire the montage and designate it as a map.

4.	On the montage overview, add a point at each location of interest (e.g., corresponding location on serial sections, or grid squares with ice of the right thickness.)  Select ‘Acquire’ for each point to set it up for automatic acquisition.

5.	At one of the locations of interest, draw a polygon around the area desired for a medium-mag map.  Go to an intermediate mag (like 3000x) so that a map of this area will be a reasonable number of pixels.  Select ‘Setup Polygon Montage’ to define a montage area.

6.	Acquire a montage to check the area.

7.	If necessary, shift the registration.  Add a point on an identifiable feature on the low-mag map and make it a registration point.  Increment the current registration number and mark the same position on the new image as a registration point with the same number.  Select ‘Transform Items’.

8.	Select ‘Acquire Areas’ to acquire all of the areas automatically.  Go do something else; these maps may take about 5 minutes each.

9.	When you load an individual map after it has been acquired, you can simply click in the map then press ‘Go To Marker’ to move the stage to the selected location.  At this point you could acquire a tilt series at high mag.

10.	Close the montage file used to store the maps before trying to start a tilt series.

If you want to find corresponding points after rotating a grid by 90 degrees, you could use the following procedure:

1.	Move the stage to four well-defined points (e.g., the corners of a section of interest), add the stage position as a Navigator point, and make each one a registration point.

2.	Add a point at each location of interest (i.e., each place where a tilt series is taken.)

3.	Rotate the grid.

4.	Increase the current registration number to 2.

5.	Find the four registration points and add each one as a registration point in registration 2, making sure that the registration point numbers correspond to the first registration.

6.	Select ‘Transform Items’ in the Navigator menu.

7.	Select one of the points of interest in the Navigator list box and press ‘Go To XY’.  You should be close to the desired point.  Errors of 5-10 microns are likely.

Large maps are memory intensive.  You should use binning (e.g., to 1Kx1K) to make them more information-dense.  If you keep many of them around by copying them to other buffers, you can easily run out of memory and start using swap file space.  The memory use of maps is minimized by an option (in the Navigator menu) to convert maps to bytes, which is on by default.  With this conversion, a map will typically occupy as many bytes as pixels (e.g., a 10K by 10K image will require 100 MB).  Without the conversion, a map will take 3 times as much memory because the image is stored as 2-byte integers and an additional byte array of the same size is needed for display.  You might need to turn off the option if the byte conversion is truncating the intensities inappropriately so that you cannot see light or dark details.  If this occurs, turn off the conversion, read in the map, and type new values in the White or Black text boxes of the Image Display control panel to keep the desired intensities from being truncated.  Be sure to turn the option back on.

If performance starts to suffer, use the Task Manager to assess the memory and swap file usage.  To recover the memory occupied by a map in a higher buffer, copy a small image to that buffer.  If you are going to use the Navigator extensively, get as much memory as possible for the Tecnai computer and make sure the swap file space is large (1 GB).


Setting Up SerialEM

Program Installation

A Minimal Setup Procedure

Starting with a Generic Properties File

Initial Camera Setup

Checking the Magnification Table

Tuning Gatan Camera Timing

Optional Tuning of Camera Timing

Measuring Tietz Camera Timing

Setting up Tietz Camera Binning Offsets

Calibrating Image Shift and Pixel Size

Focus Calibration

Other Microscope Calibrations

Setting Up X Ray Removal from Dark References

GIF Calibrations

GIF Properties

Final Tasks

Program Installation

SerialEM should be installed in a folder C:\Program Files\SerialEM.  After you unpack a version of the program and the ‘framework’ and ‘tools’ packages, this folder should start with the following files and folders:

SerialEMproperties.txt 	- the Properties file, read by SerialEM, edited by hand

SEMsystemSettings.txt	- a default settings file for users that do not have settings yet

Admin	- a subfolder with working copies of files for administering the program

SerialEM_2-x-x	- a subfolder with files for one version of the program

Tools	- a subfolder with sample files and other tools for setting up the program

The SerialEM_2-x-x folder should contain the following files:

SerialEM.exe	- the executable

SerialEMCCD.dll	- the plugin to DigitalMicrograph

SerialEMCCDps.dll	- the proxy-stub DLL for communicating with SerialEMCCD

register.bat	- a batch file for copying the plugin and registering both DLL’s

SerialEM.HLP	- the main help file

SerialEM.cnt	- help contents file

When you first install the program, or when you get a new version, copy (not move) all of these files into the upper folder (SerialEM).  If you have a Gatan camera, make sure that DigitalMicrograph is not running, then run register.bat by clicking on it.  This will copy SerialEMCCD.dll to the folder

   C:\Program Files\Gatan\DigitalMicrograph\Plugins

 and register both of the DLL’s.  After you do this, find the copy in that folder and check its properties to make sure that it is readable by users.  You can use this same procedure to revert to a previous version if necessary.

The Admin subfolder contains a working copy of the Properties and system settings files and the Calibrations file (SerialEMcalibrations.txt, which is read and written by SerialEM.  It also has a version of SerialEMsettings.txt that will make the program use these working copies, and a shortcut that will start the program in this folder.

If you did not get a ‘framework’ package already customized for your scope, the Properties files in both the SerialEM and the Admin folders are incomplete and should be edited to incorporate camera and magnification information, as indicated in Starting with a Generic Properties File

Make sure that all files in the installation folder (SerialEM) are readable by users and that users can write to this folder, which is the default location for the gain references to be placed.

Users should start SerialEM by means of a shortcut on their desktops.  After creating the shortcut, always edit its properties to start in a folder belonging to the particular user.  That way, each user will have their own settings file, separate from the one that inevitably ends up in the SerialEM folder because someone starts SerialEM by clicking on the program itself.

Note that a user’s settings file contains an entry for the system path.  A user can change this to point to a different location where the program will seek the Properties and Calibration files.  This provides two possibilities: that the program can be installed in a folder other than the default one; and that a different set of properties and calibrations can be set up for a different operating voltage.  It also provides for the arrangement described next.

As the program administrator, you should start SerialEM with the shortcut in the Admin subfolder (copy this shortcut to the desktop if you want.)  When you start SerialEM with this shortcut, you will access only the files in this folder.  Use this shortcut and edit the Properties file in this folder when you are first setting up the program or later if you are redoing calibrations or experimenting with properties.  Then when you are done, copy the Properties and Calibration files up to the SerialEM folder for general use.

Whenever you start the program to do some calibrations, turn on ‘Administrator mode’ (in the Calibration menu).  In this mode, you will get extra output from some calibration procedures, and you will also be protected from exiting without saving calibrations.

The procedures below refer to a number of entries to be made or modified in the properties file.  Each entry has a mixed case keyword followed by one or more values.  See Property File Entries for more details.

A Minimal Setup Procedure

Do you find the full setup procedures daunting?  Do you want to do just enough to get the program usable and try taking a tilt series?  If so, you can do the setup in two stages, first going through all of the items indicated as Priority 1 below, then going back later and doing Priority 2 items.  (It is strongly recommended that you not stop with Priority 1 items.)  When you read the explanation of some of the Priority 2 items, you may find that they are not relevant to your needs and can be skipped.  In addition, there are some procedures documented below “for the sake of completeness” which you can almost certainly skip.

The initial minimal procedure would be:

1.	If you started with a generic Properties file, insert appropriate camera and magnification table entries (Starting with a Generic Properties File).

2.	Adjust the camera settings in the Properties file (see Initial Camera Setup ), paying particular attention to ‘GainNormalizeInSerialEM’, the first 3 items in the general camera parameter section, and the parameters for each camera.

3.	If you did not get an initial Properties file appropriate for your voltage and configuration (GIF versus nonGIF), you need to check and set up the magnification table (Checking the Magnification Table ).

4.	Run a DigitalMicrograph script and a procedure in SerialEM to set the camera timing (Tuning Gatan Camera Timing )

5.	Calibrate image shift from the lowest M-mode magnification to the highest mag at which you want to try the program, following the procedures in Calibrating Image Shift and Pixel Size indicated by “if you just want to get the program running”.

6.	Calibrate autofocus (Focus Calibration ).

7.	If you have a GIF, calibrate magnification-dependent energy shifts (GIF Calibrations ).

8.	Perform the tasks in Final Tasks .

After doing these items, you can work with the program and try doing a tilt series, but you will not be able to control the intensity during a tilt series unless you calibrate beam intensity for at least one spot size (see Beam Intensity ).

Starting with a Generic Properties File (Priority 1)

If your properties file has not been configured for your scope yet, you should edit the generic file that has been supplied to incorporate camera and magnification information.

1.	At the place in the file that says ‘INSERT CAMERA PROPERTIES’, insert text from the most appropriate camera properties file in the Tools folder.  Do this for each camera, increasing the number on the first line (‘CameraProperties’) by 1 for each camera.

2.	At the place in the file that says ‘INSERT MAGNIFICATION TABLE’, insert text from the most relevant magnification table file in the Tools folder.  If none is relevant, then follow the procedure in Checking the Magnification Table when you get to that point in the procedures.

3.	Search for the text ‘SET TO’ and change the values as appropriate for the KV or other features of your scope, as indicated in the comments.  The values for 200 KV can be used for 120 KV.

Initial Camera Setup (Priority 1 and 2)

You should set the basic properties of your camera(s) in the properties file before starting SerialEM.  If you received a file configured for your scope, most of this should be set up already, but you should go through these lists of items to find the ones you do need to change and to become more familiar with the intricacies of camera setup.  If you are going to set GainNormalizeInSerialEM to 0, you do not need to specify UsableArea, BadColumns,or DMGainReferenceName.  However, gain normalizing in SerialEM has several advantages, including faster image acquisition, the ability to retain many dark references, and the ability to remove X rays from dark references.  (DigitalMicrograph now maintains only one dark reference per binning, which will force a lot of unneeded dark reference acquisitions when switching between parameter sets.)

General camera related parameters (and a few other things that could be dealt with later, but you might as well attend to them now or be aware of them.) (First 3 are Priority 1):

•	MRCHeaderTitle: Set this to the title that you want to appear in your output MRC files

•	NumberOfCameras: Set this to the number of cameras listed in your properties file

•	ActiveCameraList:  List the camera numbers in the order in which the cameras appear in DM.  The camera number is the number on the ‘CameraProperties’ line, numbered from 0.

•	ReferenceMemoryLimitMB:  This number, in MB, determines how many gain and dark references can be held in SerialEM at once.  160MB should be adequate for a 4K camera.  If your system does not have much memory and you have only a 2K camera, the number can be smaller (50MB).

•	GainRefInactivityLimit:  This parameter tells SerialEM how often to check for new gain references in DM.  It should be shorter than the minimum time that it would take for a user to take a good gain reference in Digital Micrograph.  If you have a 1K camera, 60 would be appropriate.

•	DarkRefAgeLimit: Set this time limit (in seconds) smaller if your camera needs new dark references frequently.

•	DefaultCameraDivide16BitBy2:  This parameter sets the default behavior with 16-bit cameras; set it to 1 to have 16-bit images divided by 2.  Dividing by 2 is appropriate when the last bit is noise, and will eliminate the problem of how to store 16-bit data.  Users will have their own individual settings that they can change, but this property determines their initial setting.

•	DefaultActAfterExposures:  If this parameter is 1, then by default the program will perform actions during camera readout, such as tilting and changing image shift.  This has worked flawlessly in Boulder but seemed to fail once at Scripps.  If you ever get a tilt series with two images at the same tilt after a skipped tilt, this feature has failed and you should set this property to 0.  Do not try to use this feature with a Tietz camera until the camera timing has been determined.

•	BeamCalMaxCounts: The beam intensity calibration procedure may fail if this value is not appropriate for your camera.  Set it to about 2/3 of the saturation limit for your camera.  For a 16-bit camera, specify this in original counts before division by 2.

Parameters for each camera (all Priority 1):

•	Name: Pick a name for your camera that users will recognize.

•	DMGainReferenceName:  find the gain reference in the DM Reference Images folder and copy the name exactly as it appears.

•	Set CameraSizeX and CameraSizeY

•	UsableArea: Insert the coordinates of the last good pixels on the edge, which are one pixel in from the rows and columns listed in the DM defect table.

•	BadColumns:  Uncomment if there are any bad columns and list them, separated by spaces, using the same numbers as in the DM defect table.

•	Binnings: List the binnings that you know work and that you want the users to see (the supplied default should be OK).

•	MakesUnsignedImages: set to 1 for a 16 bit camera.

•	BeamBlankShutter: Should be 1 if your scope is configured typically, but needs to be 0 if your beam shutter is connected to the standard instead of the alternate shutter.

•	SetAlternateShutterNormallyClosed:  Set this to 0 if for some reason you do not want SerialEM to set the beam shutter normally closed for this camera upon startup.  Note that if your beam shutter is connected to the standard instead of the alternate shutter, this setting will still do what you want.

•	OnlyOneShutter:  Insert a line setting this to 1 if the camera is connected to only one shutter.

•	If you have DigitalMicrograph 3.7.1.5, uncomment ‘CanDriveDMBeamShutter 0’ and ‘CanDriveDMSettling 0’; otherwise, the plugin should be able to determine these capabilities.

•	Timings will be dealt with later.

•	Check the settings for Retractable, GIF, and Order for appropriateness.

•	If you have a GIF, you will probably want ‘InsertTVToUnblank’ set to 1, as it allows both cameras to be maintained in the configuration of Alternate Shutter Normally Closed without special hardware.

•	Set PixelSizeInMicrons

•	Set CountsPerElectron if you have a good estimate of the camera gain.  These would be counts before division by 2 for a 16-bit camera.  The program needs this value to compute electron dose.  It also needs beam intensity calibrations and spot intensity calibrations.  In addition, a daily dose calibration is required; this can now be done automatically when taking the gain reference.

•	Leave ‘RotationAndPixel’ lines, if any, commented out.

For a Tietz camera, attend to the following settings:

•	Set TietzCameraType to 2 for PXL camera, 3 for PVCam camera (PVCAM driver using ICL), 4 for FastScan camera using MVTitan frame grabber, 5 for SCX camera (PVCAM without ICL), and 6 for FastScan camera using FireWire 1394 interface.

•	Set BuiltInSettling to 0.

•	Set MinimumDriftSettling to 0.01.

•	ExtraBeamTime and StartupDelay will be set from a timing calibration.

•	Set TietzCanPreExpose to 1 if the camera has a shuttering mode that allows pre-exposure of the specimen, using the film shutter and beam blanker.

After you have checked out these settings, you are finally ready to run SerialEM and try taking some pictures.  Experiment with the different camera parameter sets, varying the binning, the area being acquired, and the shuttering selection if appropriate.  You may have to take a gain reference in SerialEM before you can get a gain normalized image.  If you run into problems, try taking unprocessed and/or dark subtracted images as well as varying the binning and image area to see under which conditions the problem occurs.

At this point you may discover that the control panels on the left are not all the same width, and that some text appears truncated in the control panels or dialogs.  This happens when the display is not running at 120 dpi, which was the Tecnai standard early on.  To solve this problem, add a property file entry “DisplayIsNot120DPI   1”

Checking the Magnification Table (Priority 2)

You should start with a properties file that has a magnification table but does not have any ‘RotationAndPixel’ lines in it.  Start SerialEM and run the ‘List Mags’ command from the ‘Calibration’ menu.  Compare this listing with the magnification table in the properties file.  If it matches, you are ready to go on.  If not, you need to cut and paste the output of ‘List Mags’ into the properties file.  Then step through the mags with the screen down and type in the mag that the Tecnai shows.

Now that you know the relation between mag index and mag, find out which is the lowest mag in M mode and verify that LowestMModeMagIndex is correct.

If you have a GIF, repeat these steps with EFTEM mode on – i.e., check the values in the mag table from Boulder, and correct them if necessary.

Tuning Gatan Camera Timing (Priority 1)

Camera timing settings are important for proper function of SerialEM’s ‘Dual shuttering – minimum exposure’ option, which is needed to minimize specimen exposure using the standard shutter and to provide a flexible range of drift settling times.  Measuring them involves two steps: using a DigitalMicrograph script to estimate the BuiltInSettling, or clear time of the CCD chip; and using a procedure within SerialEM to determine the average delay for an exposure to start after SerialEM issues a request for one, and the variability in this delay.  An alternative procedure for measuring the clear time exactly with an oscilloscope is described below, but this should not be necessary because the delay measurement will compensate for inaccuracies in the clear time estimate.

If the camera controls only one shutter, this procedure is not so important but it can still be run to provide a check on the timing for performing tasks after an exposure.

To estimate BuiltInSettling, open the ‘cleartime.s’ script provided with the ‘Tools’.  You can do this with the File - Open menu entry in DM, or just by clicking on the file.  Run the script by pressing Ctrl-Enter.  It will time a series of exposures and report the mean time.  It will then recommend values for BuiltInSettling based on subtracting 0.155 second for large format one-port readout cameras (Megascan 795 and Ultrascan 890, 2Kx2K 30-micron pixel or 4Kx4K 15-micron pixel) or 0.065 second for the four-port readout Ultrascan 895.  These correction factors are based on the 4 cameras that I have been able to run the script with.

If you have more than one camera, change the ‘cameraNumber’ value at the top of the script and rerun it.

Once you have entered a correct value for BuiltInSettling in the properties file, restart SerialEM.  Change the ‘Trial’ camera parameters so that the exposure time is 0.4 seconds and set the binning to get a 1024x1024 pixel image.  Take a ‘Trial’ picture without a specimen and adjust the beam to give an image with moderately high counts (between 1/3 and ¾ of camera saturation).  Then select Camera Timing  in the Calibration menu and accept the default entries.  This routine will take 100 exposures with timing parameters that will allow it to estimate the delay time for the exposure to start from the image intensity.  It will recommend values for StartupDelay, MinimumDriftSettling, and ExtraBeamTime, and it may also recommend a change in BuiltInSettling to keep StartupDelay from being negative.  Insert these in the properties file entries for the particular camera.  If you have more than one camera, repeat the procedure with the other camera.

You should rerun the camera timing procedure and adjust your properties entries if there is a computer upgrade, a major software upgrade, or the addition of a GIF.  The timing also seems to depend on whether images are gain-normalized in DM or in SerialEM, so rerun the procedure if you change the GainNormalizeInSerialEM property.

Optional Tuning of Camera Timing (For the Sake of Completeness)

If you want to measure the BuiltInSettling exactly, you need a two-channel oscilloscope, two BNC cables, and two BNC tees.  Connect the standard and alternate shutter outputs on the camera controller to the two scope inputs.  Display the alternate shutter above the standard one.  The alternate shutter is high (5 V) when it is closed and drops to open.  The standard shutter behaves the same for an upper camera and is inverted for the GIF shutter, i.e. low when closed then high when open.  Set up for triggering on the downward phase of the alternate shutter.  In DM, take a picture with standard shuttering and 0.5 second exposure, then take another, which will not require a dark reference.  You should get a pattern similar to the first picture shown in  Image Acquisition , except that the traces will be upside down.  First the beam shutter opens while the standard shutter closes; then the standard shutter opens again for the 0.5 second duration.  Your goal is to measure the time between the opening of the beam shutter and the re-opening of the standard shutter to within 20 ms. (Note that if you have the standard shutter connected to the beam shutter and the camera configured to have standard shutter normally closed instead of alternate shutter normally closed, you will see the same behavior but with the two traces swapped.)

The ExtraUnblankTime is relevant only if beam shutter control is broken (DM 3.6.0 – 3.7.1.5).  It would be adjusted by taking 0.1 sec exposures with ‘DM film shutter’ (which will have constant means) then taking exposures with ‘Dual shuttering’ (which will vary).  The ExtraUnblankTime is adjusted to make the ‘Dual shuttering’ exposures have the same mean, on the average, as the ‘DM film shutter’ exposure.

The ExtraOpenShutter time can be assessed with the oscilloscope set up as described above.  Set this entry to 0 and restart SerialEM.   Set up an exposure with ‘Dual shuttering’ and drift settling longer than the clear time, e.g. 1.3 seconds.  Watch the shutters during an exposure.  The beam shutter will open more than 1.3 seconds before the film shutter – the amount extra is the ExtraOpenShutterTime.

Measuring Tietz Camera Timing (Priority 2)

Calibration of the timing of a Tietz camera is needed only to allow the program to takes actions (e.g., tilt and image shift) during image readout.  Change the ‘Trial’ camera parameters so that the exposure time is 0.4 seconds and set the binning to get a 1024x1024 pixel image.  Take a ‘Trial’ picture without a specimen and adjust the beam to give an image with moderately high counts (between 1/3 and ¾ of camera saturation).  Then select Camera Timing  in the Calibration menu and accept the default entries.  This routine will take 100 exposures with timing parameters that will allow it to estimate the delay time for the exposure to start from the image intensity.  It will recommend values for StartupDelay and ExtraBeamTime.  Insert these in the properties file.  If the procedure measures a delay successfully, then you can DefaultActAfterExposures to 1.

Setting Tietz Camera Binning Offsets (Priority 2)

The first Tietz camera that SerialEM was run with had a problem with gain-normalization of binned images.  The problem is solved with ‘BinningOffset’ entries in the camera properties section.  These entries are determined empirically by changing the offset values in the Properties file and stopping and restarting the program.  Here is a procedure, written up from dim memories of adjusting the offsets:

1.	Take a fresh gain reference, unbinned.

2.	Take a dark-subtracted then a gain-normalized image at binning 2.  Toggle between the two to see if the fixed pattern in the dark-subtracted appears in the gain-normalized image.  If so, you will usually see a (subpixel) shift in the pattern.

3.	Insert an entry like ‘BinningOffset 1 2 1 1’ in the Properties file.  The first ‘1’ refers to the binning of the gain reference, the ‘2’ refers to the binning of the image, and the next two numbers are the offsets in X and Y, which can be small positive or negative numbers.  Unfortunately, I don’t remember the correspondence between the direction of shift and the sign of the needed offset.  If the image appeared to move only in one direction, use a zero for the other direction.

4.	Restart SerialEM and take dark-subtracted and gain-normalized images again.  If the shift is worse in a direction, invert the sign of the shift; if the shift is less but still present, increase the shift.  Repeat this procedure until the image looks clean.

5.	Repeat the procedure at all higher binnings and insert ‘BinningOffset’ lines as needed.

6.	Take a gain reference with binning of 2.

7.	Take a dark-subtracted then a gain-normalized image at binning 4.  Insert a line like ‘BinningOffset 2 4 1 1’ and adjust the offsets to get a clean image.

8.	Repeat the procedure for all higher even binnings.

Binning offsets are also needed for Gatan 4-port readout cameras but these offsets are predictable and the program will set them up automatically.

Calibrating Image Shift and Pixel Size (Priority 1 and 2)

The procedure described next involves calibrating the image shift at a series of magnifications and saving that information in the calibrations file, as well as taking a series of images and measuring the magnification and change in image rotation directly from them and entering this information into the properties file.  In principle, this is redundant information, because if SerialEM has a calibration of image shift at all relevant mags and a pixel size and true rotation of the tilt axis at one mag, it can use that calibration to determine pixel size and image rotation at all of the other magnifications.  Alternatively, it can use information about pixel sizes and relative rotations between mags to derive an image shift calibration, although this would be less accurate.  If you want to trust the image shift calibrations, you could calibrate image shifts at a range of mags and measure just one or two pixel sizes, as indicated below.  Finally, if you want to do just enough to get the program running, you can calibrate a restricted range of mags and skip saving images.

You may also want to calibrate image shifts in Low Mag mode.  This is needed in order to use the Navigator to make low mag maps.  If you want to do this, you need to measure one pixel size in Low Mag mode as well and measure the rotation between regular and Low Mag.

On a JEOL, the beam is not moved along with the image, so SerialEM needs to apply beam shift whenever it does an image shift.  This will not happen until you do a beam shift calibration, which needs to be done after at least one image shift calibration.  Thus, you need to spread the beam to at least twice the size of the CCD camera to get through the image shift calibrations successfully.

To calibrate magnifications and image shifts, do the following:

1)	Place a standard cross-line grating in the scope (2160 lines per mm).  I prefer the kind with latex spheres, which are easier to focus.

2)	Make sure your chosen field of view is eucentric.

3)	Use a moderate objective aperture like 40 microns for contrast.

4)	Turn on intensity zoom for convenience.

5)	Turn off “Align on Save” in the ‘Buffer’ control panel (in the options section).

6)	First go to a moderate mag like 15,000 and focus there.

7)	Set up the Trial parameter set to take 512x512 images, and exposure time as well as the beam strength to get a nice clean picture.

8)	Turn on Administrator mode and open a Log Window.

9)	Calibrate image shift from scratch at that mag (see IS from Scratch  and Image Shift Calibration  in the Calibration menu).

10)	Open a new file to save images to (skip this if you just want to get the program running).

If you want to do calibrations in Low Mag mode, first go to the highest LM-mode magnification.  Focus as well as you can, and then calibrate image shift from scratch.  Save the last image to the file, or take another Record after adjusting intensity if desired.

Next go to the lowest M mode magnification and do the following for each mag up to as high as you think is needed:

1)	Focus carefully (minimum contrast with aperture out).

2)	Insert aperture.

3)	Calibrate image shift.

4)	Save the last image to the file, or take another Trial after adjusting intensity if desired.  If you don’t want to measure pixel size and rotation at each mag, just save the image at the lowest M mode mag.  (Skip this if you just want to get the program running).

Be sure to save your calibrations (in the Calibration menu).  You are done with this section if you just want to get the program running.

Open a copy of the mag calibration spreadsheet.  Set the binning and the size of the pixels on the CCD to the correct values.  If necessary, adjust the magnifications listed in the first column.

Further processing is done in IMOD as follows.  Load the stack of grating images into 3dmod with:

3dmod filename.st filename.mod

On each image, draw one contour with 4 lines (5 points), going along the grid lines and enclosing about as big a rectangle as you can.  Make the last point coincide with the first.  Count the number of squares in the two directions and insert the total number of squares around the perimeter into the spreadsheet in the ‘Model Grid lines’ column.  Use Edit-Contour-Info to find the length of the contour, or run imodinfo to get values for all contours after saving the model.  Insert these values into the ‘Contour Length’ column.  You will see the pixel size in the ‘Unbinned cal pixel’ column.  The formula for the pixel size is (463 nm/square * number of squares) / (number of pixels * binning).  Draw contours on the images until you reach a mag where you can no longer enclose 4 squares.

If you only saved one image, you have the pixel size and you are done.  You would now enter one line in the properties for this camera, e.g.:

RotationAndPixel  17  999  999  4.95

Where 17 is the mag index, the two 999’s indicate that relative and absolute rotation were not measured, and the 4.95 is the pixel size.  Skip to the end of this section to see how to enter the absolute tilt axis rotation.

If you have more than one image, next use Midas to get a rough prealignment of the images:

midas filename.st filename.inixf

For each image pair, change the magnification (Ctrl-right mouse button) so that they are close to matching, and shift and rotate as necessary to get into rough alignment.  As long as they are aligned in the center, xfalign should be able to complete the job.  If you can’t match up a pair of images because the microscope is not well enough aligned, just skip the pair.  Save the transforms and run:

xfalign –red 0 –bi –ini filename.inixf filename.st filename.xf

When it is done, restart Midas with:

midas filename.st filename.xf

Look at each pair to make sure it is well aligned.  If you only have two images, you can skip the spreadsheet entries and proceeding to putting two lines in the properties for this camera, e.g.:

RotationAndPixel  16  999  999  10.5

RotationAndPixel  17  158.17  999  9.90

Where 10.5 and 9.90 are the pixel sizes measured for the two mags, 158.17 is the NEGATIVE of the rotation angle shown in Midas, and the 999’s indicate unmeasured values.  Skip to the end of this section to see how to enter the absolute tilt axis rotation.

If you took a full set of images, copy the Midas rotation, magnification, and stretch values into the appropriate lines of the spreadsheet (the first image pair provides data for the second mag).  The rotation is just there to keep a record of it, but the Midas mag and stretch are resolved into a single number, in the ‘Midas delta mag’ column.  For the lower mags, you can compare these with the ‘Delta pixel’ values to see how accurate these two methods are.  The real use of the delta mag’s is to derive pixel sizes above the last mag where you could measure it directly.

Suppose the last mag where you measured the pixel (or the last one where you trust the measurement) is on line 11.

1)	Put a 1 on that line in the ‘Cumulative delta mag’ column (cell J11).

2)	Put a formula in the cell on next line to multiply the cell above by the ‘Midas Delta mag’ on that line (J11 * E12 in J12), or just copy this formula up from the cells below.  Either way, fill the cells below with this formula.

3)	In the ‘Derived pixel’ column, put a formula to multiply the last measured pixel value by the cumulative delta mag on that line (H$11 * J11 in K11).  Copy this formula down to the cells below.

4)	The ‘Unbinned pixel’ column takes its values from the ‘Unbinned cal pixel’ column for low mags and from the ‘Derived pixel’ column for high mags.  Copy formulas down from the top or up from the bottom so that all of the values in this column are based on the correct pixel size.

5)	The film magnification and unbinned and binned pixel sizes are copied to a table below the main one.  Adjust the formatting of the cells in this lower table to show the desired number of significant digits.  Print this portion of the spreadsheet and post it for users.

6)	The last column, ‘Ratio’, shows the magnification from film to camera.  Average these numbers, or leave a few out if they appear out of line, to obtain ‘FilmToCameraMagnification’.

Enter the appropriate numbers from the spreadsheet in a series of ‘RotationAndPixel’ lines in the properties file.  Each line should have:

RotationAndPixel  <Magnification index>   <the NEGATIVE of the Midas rotation>  999  <Pixel size>

The last number missing from the RotationAndPixel entries is an absolute calibration of the rotation of the tilt axis.  This is most reliably obtained from a fiducial alignment of a tilt series.  Once this value is available, replace the 999 on the RotationAndPixel line for the relevant mag with 90 degrees plus this value.  If you only measured one or two pixel sizes, this angle will most likely be measured at a different mag, in which case you would add a line such as:

RotationAndPixel  23  999  77.5  999

Where 77.5 is the measured absolute rotation.

You can also use the rotation angle from a tilt series alignment to adjust the entry for ‘GlobalExtraRotation’.  On the Tecnai, if the value from alignment is near 0 then use it directly; if it is near +/-90, then adjust it by 90 degrees to be near 0.  On a JEOL, the basic principle is that the sum of the rotation angle in the magnification table for this mag (which could be wrong), plus the GlobalExtraRotation, should equal the absolute angle on the RotationAndPixel line.

Focus Calibration (Priority 1)

Focus only needs to be calibrated at one magnification as long as there are image shift calibrations available to transfer the focus calibration accurately to other mags.  Pick a mag in the range where people will work (~20K).  Set up the Focus parameter set to give moderately good, quick pictures.  Wide quarter unbinned, and center half binned are two possibilities; if you have a smaller format camera (e.g., 1K  24 micron pixels or 2K 15 micron pixels, use a center half area).  Defocus by 10 microns and run ‘Report Shift & Drift’ in the Focus menu.  Adjust the beam tilt so that this procedure reports a total shift (combining X and Y shifts by the square root of the sum of squares) equal to ~1.5% of the camera field for a large format camera or ~3% of the field for a small format camera (i.e, about 30 unbinned pixels for a large-pixel camera (24-30 microns) or about 60 unbinned pixels for a small-pixel camera (14-15 microns).  Restore the defocus to zero and start the focus calibration procedure (see Autofocus  for more details.)  Accept the defaults to calibrate a nice big range of defocus.

Other Microscope Calibrations (Priority 2)

Low Magnification and Stage Calibrations:  These calibrations are needed to do montaging with stage movement and to use the Navigator to acquire low and medium-mag maps.  For these, you will need a specimen that is moderately rich in detail and that is either on a slot grid or on a grid with a very large mesh.  Start at 1000-2000x and find an area with good features.  Focus; if you are in LM mode, try to find a consistent focus that might correspond to what people would actually focus at in low mag mode.  Run the Stage Shift  command from the Calibration menu.  If you did not yet calibrate image shift at this magnification, then run an image shift calibration also.  Repeat this procedure at the next lower 3 or 4 mags (i.e., get a total of 4-5 stage shift calibrations), then just do image shift calibrations down to the lowest mag that might be used for mapping.  You may also need to do an image shift calibration at mags between the one you started at and the highest LM mode mag.

Beam crossover calibration:  This calibration is needed if you want to have intensity calibrations on both sides of crossover.  Go to a high magnification like 100K, select Beam Crossover  in the Calibration menu, and adjust the beam to crossover at each spot size.

Beam intensity calibration:  This calibration is best done with no specimen.  The procedure must be done for each spot size that will be used for tilt series; if low dose is going to be used, this might mean spots up to 6 or 8.  In the latter case, try to calibrate up to whatever spot size gives 0.3 nanoamp of current.  Start the procedure one or two steps above the highest mag that might be used (~100K for low spot size numbers, lower mags for the spots that will only be used for low dose?).  An intensity calibration will work on only one side of crossover.  You can calibrate one side of crossover based upon the users’ most common practice.  Alternatively, you can calibrate both sides of crossover provided that you have done the beam crossover calibration.  See Beam Intensity  for more details.

Spot intensity calibration:  This calibration measures the relative intensity of the spots and is needed to allow an electron dose calibration at one spot to be transferred to other spot sizes.  It should be done at an intensity setting that is within the calibrated range for all spots of interest.  It is best done with no specimen.  As with the beam intensity calibrations, you can do in one side of crossover, or on both.  See Spot Intensity  for more details.

Beam shift calibration (Priority 1 on JEOL):  This calibration allows the user to shift the beam by a controlled amount by placing a point on an image.  It is essential on the JEOL for getting the beam moved correctly when image shift is used.  Separate calibrations can be done in regular and low magnification modes.  Follow the procedure in Beam Shift  .

Small screen factor:  This is needed to get the same current readout with the small screen in and out.  Fill the main screen with a moderately bright, uniform beam (5-10 na).  Note the current reported in SerialEM.  Insert the small screen.  Compute the ratio of the current now to the current on the main screen.  Go to the properties file and multiply the existing SmallScreenFactor by this ratio.  Restart SerialEM and check that the current corresponds adequately now.

There is also a ScreenCurrentFactor that requires a Faraday cup measurement to calibrate.  The screen current calibrations on Tecnais can be off by over 30%, so this is worth doing.  If you determine the ratio between the current measured with a Faraday cup and the current reported by SerialEM, then multiply the existing ScreenCurrentFactor by this ratio.

C2 factors:  This calibration is needed to make the C2 lens percentage reported in SerialEM correspond to the values shown in Tecnai User Interface.  To do this calibration, first add the C2 lens readout to your TUI workspace.  Then select Set C2 Factor  in the Calibration menu and enter the C2 value at each spot size.

Tilt axis offset:  This calibration is needed to shift the microscope image shifts so that they are centered on the tilt axis of the goniometer.  FEI service engineers should be able to adjust the tilt axis to within 1 micron of the optic axis, but if this has not been done, this setting will help to minimize movements in Y and Z when tilting.  You need a specimen suitable for running the eucentricity routines with.  Go to the center of the grid, run Eucentric - Both  in the Tasks menu, then run Eucentric - Fine  in the Tasks menu 2-4 more times.  If the numbers are consistent, enter their average as the TiltAxisOffset in the properties file.  This value has an effect only if individual users turn on ‘Center image shift on tilt axis’ in the Image Alignment and Focus control panel.  If you do calibrate this setting, check it after the stage is worked on, as it can easily change.  Note that if you have this option selected and you measure the offset with the eucentricity routine, you need to ADD the measured offset to whatever value is already present.

Setting Up X Ray Removal from Dark References (Priority 2)

The main task here is to determine the hot pixels and hot columns in the CCD so that these are not erroneously treated as X rays in a dark reference.

1)	If you have a 16-bit camera, turn off division by 2 if it is on, or remember to double the absolute criterion that you determine when you enter it in the properties file.

2)	Set up a parameter set to take full-field unbinned 3 second exposures, and select the option to take a new dark reference every time.

3)	Open column values and spread the beam a lot (or select a high spot size) so that the beam will not saturate the camera.

4)	Take an exposure, then select ‘Show Dark Ref’ from the Camera menu.  Save the dark reference to a file.

5)	Do this 3 times, saving all 3 images to one file.

6)	Remember to turn off the option for a new dark reference every time.

Transfer this stack, the program Findhotpixels, and findhotpixels.com to an IMOD-capable environment on a Linux machine.  Edit findhotpixels.com to provide the name of the image stack, the name of a model file to place points in, a threshold for hot pixel strength, and a list of any hot columns that appear in the images.  Number the hot columns from zero.  With a Gatan US 4000 camera you will need to exclude a large number of columns near the edges, 20 or more.  Enter subm findhotpixels.

Alternatively, the ‘Tools’ contain a Windows version of Findhotpixels with the necessary libraries.  You can run it interactively from the DOS command line, or take it to a Windows machine with IMOD installed and use the command file there.

You will get a list of the hot pixels and their mean values in the log file.  You can also look at the model on top of the images to verify that the program has found hot pixels.  The log file ends with a series of lines of the form:

HotPixels  X  Y  X  Y  X  Y ..

Add these lines to your properties file.

If you have any hot columns or bad columns that appear hot, add an entry

HotColumns

and list the column numbers on this line, numbered from 0.

Uncomment all of the lines about DarkXRay…  Set the DarkXRayAbsoluteCriterion to the threshold you set for Findhotpixels (but double it if images from a 16-bit camera were divided by 2).  The MaximumXRayDiameter can be set to 6 for 15 micron pixels or 4 for 30 micron pixels, unless you want to analyze the situation further.

You can assess the performance of the X-ray removal with different criteria by reading your dark reference images back into SerialEM.  After reading one, copy it to Buffer A, then select ‘Fix Dark X Rays’ in the Process menu.  You can then compare the images in A and B.  Turn on Administrator mode and select ‘Set Criteria’ in the Process menu to change the criteria.  Copy the image back to A and run the removal again.

GIF Calibrations (Priority 1)

Before even trying to calibrate mags for your GIF, you may need to improve the EFTEM alignments that were supplied by FEI so that the field of view does not shift too much when you change mags.  This is a tedious and difficult procedure but is worth figuring out how to do it well.

Once you have done that, you should do a separate calibration of image shifts and pixel sizes from the calibration on the upper camera (or at least do the image shifts and one pixel size.)  SerialEM has some fancy logic for transferring calibrations from one camera to another, but I wouldn’t rely on it except for test purposes.  Do a calibration of autofocus at one mag first, because then you will be able to use autofocus to focus when you switch between mags.  Then follow the procedure above for getting the image shift calibrations and grating pictures.

In addition, you will need to calibrate the magnification-dependent energy shifts.  Not only does this provide more convenient use of the GIF, but it is needed so that various procedures can switch to lower mag when the slit is in and not lose the zero loss peak.  Follow the procedures described in  Mag Energy Shifts .  It is best to select ‘DM film shutter with beam blanking’ to get consistent pictures, unless you are sure beam blanking is working right in your configuration.

GIF Properties (For the Sake of Completeness)

There are several properties for the GIF (see GIF properties .)  The most important to check is the BiggestGIFApertureNumber, which is used to make sure that there is no aperture in the way when images are taken.  In addition, there are properties to specify delays when inserting or retracting the slit or changing energy offsets.  These timings may be OK, but here are some notes on how they were obtained.

For testing the adequacy of the delay on removing the slit:

1.	You need to have a situation where the ZLP is misaligned by at least 10 ev.  Perhaps align the ZLP at one mag, then turn off ‘Mag-dependent energy shifts’ in the Filter Control panel and switch to another mag where the ZLP is shifted by at least 10 ev.  (This was easier before SerialEM detected ZLP alignment automatically.)  The point is that you want to be able to take a picture at zero loss with a 5 ev slit and get very little beam through.

2.	Enter low dose mode.  Set up view and record/preview to take pictures at this mag with the same intensity, and set the intensity and exposure time of view and preview to give thousands of counts. Use ‘DM film shutter with beam blanking’ to get consistent exposures.

3.	Set up record with 5 ev slit in, 0 loss

4.	Setup up view with slit out

5.	Hit preview to condition, then view to test, then Ctrl I for image mean.

6.	With the right delay, view will always come out right.  If the delay is too short, the image mean will occasionally fall short.

For testing the delay upon energy shift:

1.	Set up record/preview and view in Low dose so that both are at the same mag and have the slit in.

2.	Get the ZLP aligned and set up record/preview and view for 10 ev slit, slit in.

3.	Make sure View gives good consistent pictures, and set the loss in preview to the desired differential.

4.	Hit preview to condition, then View to test and Ctrl I for image mean.

5.	Test this for energy shifts like 10, 20, 40, 80, 120, 160, 200.

6.	If there are problems, you may need to test more, or just increase the delays.  Filter offset delay is specified by two linear equations, each having a base delay and an additional delay per ev of offset.

Final Tasks (Priority 1)

When you are finished setting things up, you need to copy files from the Admin folder up to the SerialEM folder.  But first check the settings in the system (default) settings file.  Run ‘Open’ in the Settings menu and load the file SEMsystemSettings.txt from the Admin folder.  Check each of the camera parameter sets.  If the capture areas are wrong (too big, too small, not centered), or the binnings are not appropriate for the camera, correct them.  You can change any other settings that you think appropriate.  Exit SerialEM, or just select Save in the Settings menu.  Copy SEMSystemSettings.txt, SerialEMproperties.txt, and SerialEMcalibrations.txt from the Admin folder up to the SerialEM folder.


Property File Entries

Many parameters can be set in SerialEM through the properties file.  They all have default values in the program, and in most cases the values in the properties file are the defaults.  A property is entered by having a line with a keyword followed by one or more values.  As of SerialEM 2.6.0, the keywords are case-insensitive, but they are most readable when shown in the MixedCase form that used to be required.  Values can be separated by any number of spaces or tabs.

General entries

General camera control properties

GIF properties

Properties for one camera

File options

Microscope properties

Autofocus and autoalignment correlation properties

Task operation properties

Tilt Series Controller properties

Beam intensity calibration properties

JEOL properties

General entries

SerialEMProperties	The file must start with this entry.
GainReferencePath	The path to gain references, if it is not the system path.
MRCHeaderTitle	Title to be placed in MRC files; full title is SERIALEM: ‘your title’  date-time
LogBookPathName	Full path name of a text file in which tilt series information will be logged.  The “log book” contains tab-separated entries as follows: Date/time, image filename, starting Z value of tilt series, total number of Z values in the file, full image X size, Y size, file size in MB, starting angle, ending angle, tilt increment, COS for cosine increments or REG if not, minutes for tilt series, frames for montage or 1x1 for single-frame, camera name, nominal film mag, binning, pixel size in nm, LD for lowdose or ND if not, slit width if filtered or 0 if not, energy loss or 0 if not filtered.
StartupMessage	This entry can be added to provide a message upon program startup.  The entry must be all one line but the message can consist of multiple lines, separated by \n.  The message is printed to the log window if it is reopened, otherwise to a message box.
DisplayIsNot120DPI	Set this to 1 to get the right font size on a display that is not set for 120 DPI (large fonts).
ExitOnBadScopeStartup	Set this to 1 to have the program exit if errors occur when connecting to the microscope.
StartAsAdministrator	Set to 1 to turn on Administrator mode when starting the program.
SmallFontsBad	1 was needed to display small fonts properly on a laptop.
SingleTecnaiObject	0 to allow threads to open a second connection to the Tecnai; 1 to shut down the standard connection while a thread has a connection, and reopen it when done.
SimulationMode	Set to 1 to indicate a microscope simulator is being used (enables some actions on JEOL).
DebugOutput	Set to 1 to obtain general debugging output in the Log Window.  Set to a list of key letters (e.g., JTC) to obtain additional output corresponding to the particular key letters.  Key letters are: i for image shift-related items, l (lower case L) for low dose, L for more verbose low dose, u for update items when polling, e for event reports, and J for complete listing of JEOL calls.

General camera control properties

NumberOfCameras	This property has been discontinued.
ActiveCameraList	A list of all of the camera numbers for cameras that are active, in order as they are seen by DigitalMicrograph.  The camera numbers can be from 0 to 3 and correspond to the numbers on the “CameraProperties” lines.
InitialCurrentCamera	The number of the camera that should initially be set as the current camera.  Without this entry, the program will start with the first camera in the active camera list, or the first nonGIF camera if that is a GIF camera.
GainNormalizeInSerialEM	1 to perform gain normalization and dark subtraction in SerialEM or 0 to have all processing done in DigitalMicrograph.
ReferenceMemoryLimitMB	The total memory allocated for dark references in SerialEM.  Needs to be large for a 4K camera, e.g., 160 MB.
DarkRefAgeLimit	When gain normalization is done in SerialEM, a new dark reference will be taken if one is older than this time, in seconds.
GainRefInactivityLimit	When gain normalization is done in SerialEM, SerialEM will examine whether there is a new gain reference in DM if no image has been taken for longer than the given number in seconds.  If DM gain references are being used, the new one will be fetched; if SerialEM gain references are being used, the user will be asked whether to use the newer one from DM instead.  This number should be set a little less than the shortest reasonable time that it would for a user to switch to DM and take a gain reference.
DigitalMicrographReferencePath	The path to the folder where DM keeps gain references.
InnerXRayBorderDistance	When removing X-rays from a dark reference, the mean of surrounding pixels will be computed from pixels that are at least this distance from all pixels above the criterion.
OuterXRayBorderDistance	Pixels will be used to compute mean of surrounding pixels if they are no more than this distance from a pixel above the criterion.
XRayCriterionIterations	If a patch of points above the basic criterion is too big, then criterion will be raised for up to this many iterations to try to get the brightest subset.
XRayCriterionIncrease	On each such iteration, the criterion applied is the basic criterion times (1 + iteration number x this value).
GlobalExtraRotation	The angle to add to the rotation angle reported by the Tecnai to get the actual image rotation, i.e., the rotation of the tilt axis from the X axis.
RetractCameraOnEnteringEFTEM	1 to have an upper camera retract and the beam blanked during the retraction when EFTEM mode is turned on.  This is necessary only if there is a pressure switch overriding the GIF’s blanking of the beam, because there is a delay between the time when the upper camera unblanks the beam and the time when the pressure switch allows the GIF to blank it again.
BlankWhenRetractingCamera	1 to have the beam blanked whenever any camera is retracted to unblock the path to a lower camera.
DefaultActAfterExposures	Set the starting or default value for the Post-actions setting that allows tilting and image shift to start during camera readout; i.e., 0 to disable post-exposure actions until the user enables them.
DefaultCameraDivide16BitBy2	Set to 1 to have all images from 16-bit cameras divided by 2 as they acquired in SerialEM.  This is a default behavior that can be changed by each user.
StartCameraInDebugMode	1 to turn on debug mode as soon as the connection to the camera is initialized.  This will print all communication with the camera plugin to the DM Results window and also provide some verbose output to the SerialEM Log Window.
TietzFilmShutterSwitchTime	Seconds to wait after switching the Tietz camera to film shutter mode; this time specifies the minimum pre-exposure time that will occur on the first acquisition after switching to film shutter.

GIF properties

BiggestGIFApertureNumber	The number of the aperture that must be in place for imaging.  You could image an aperture by entering its number here.
MinLowMagSlitWidth	Minimum slit width when doing low mag tasks.
SlitWideningSafetyFactor	When doing a low mag task, the slit will be opened if necessary to the minimum slit width, and intensity will be reduced by the ratio of original to new slit width times this additional safety factor.
FilterSlitInOutDelay	Delay time in DM ticks (60th of second) when inserting or retracting slit
FilterOffsetDelayCriterion	For filter offset changes less or more than the criterion, delay time in ticks is computed as change * slope1 + base1 or change * slope2 + base2, respectively
FilterOffsetDelayBase1	Base delay (in ticks) for offsets less than the criterion
FilterOffsetDelaySlope1	Additional delay per EV of offset for offsets less than the criterion
FilterOffsetDelayBase2	Base delay (in ticks) for offsets greater than the criterion
FilterOffsetDelaySlope2	Additional delay per EV of offset for offsets greater than the criterion
NoSpectrumOffset	Set to 1 to indicate that Spectrum Offset cannot be set and Energy Shift should be set instead.

Properties for one camera

(all times are in seconds)

(For a Tietz camera, StartupDelay, MinimumDriftSettling and ExtraBeamTime are the only timing parameters that are relevant, and any other property that refers specifically to Gatan camera or DigitalMicrograph (DM) is not relevant.)

CameraProperties	This entry, followed by a camera number from 0 to 3, signals the start of a section of properties for a camera, which can include any of the entries in this table.
Name	The name of the camera, which will appear in the camera, montaging, and tilt series setup dialogs.  Spaces are allowed.
TietzType	0 for a Gatan camera, 1 for Tietz simulation camera, 2 for PXL camera, 3 for PVCam camera (PVCAM driver using ICL), 4 for FastScan camera using MVTitan frame grabber, 5 for SCX camera (PVCAM without ICL), and 6 for FastScan camera using FireWire 1394 interface.
TietzCanPreExpose	Set this entry to 0 or 1 to indicate whether a Tietz camera has a shuttering mode that allows pre-exposure of the specimen, using the film shutter and beam blanker.
TietzGainIndex	Gain index to use for a Tietz camera.  This is not the gain itself but a number between 1 and the number of gains available.  The default is 1.
DMGainReferenceName	The full name of this camera’s DM gain reference, including spaces and .dm3.
CameraSizeX	Number of pixels in X
CameraSizeY	Number of pixels in Y
XMustBeMultipleOf	This entry constrains the size in X of images to be acquired to be a multiple of the given number.
YMustBeMultipleOf	This entry constrains the size in Y of images to be acquired to be a multiple of the given number.
FourPortReadout	Set this entry to 1 for a 4-port readout camera.
BasicCorrections	This entry determines the corrections done for all image acquisitions in DigitalMicrograph.  It can be the sum of 1 for defect correction, 16 for bias correction, and 32 for linearization.  The default value if none is entered is -1, which means that default corrections will be done for the particular camera.
MakesUnsignedImages	Set this entry to 1 for a 16-bit camera that produces unsigned 16-bit integer images.
CountsPerElectron	The camera gain, i.e. the number of counts per primary electron.  This entry is needed for dose estimates.  It should be in counts before dividing by 2 for a 16-bit camera.
Binnings	A list of valid binnings for this camera.  Up to 9 binnings may be listed and will appear in the camera setup dialog.
BinningOffset	This entry may be needed to make gain normalization work well in binned images when gain normalization is being done in SerialEM.  Follow the keyword with 4 numbers: the binning of images being normalized, the binning of the gain reference (1 or 2), the offset in X (a small positive or negative number), and the offset in Y.  Offsets may be needed for Tietz cameras.  The appropriate offsets will be set up automatically for 4-port readout cameras for binnings that are not a power of 2, but these entries could be used to override the assumed settings.
UsableArea	Specifies the coordinates of the usable area on the CCD, in Digital Micrograph coordinates numbered from 0.  Follow the keyword with the top, left, bottom, and right coordinates of the last good column or row on the chip.  This is needed if gain normalization is being done in SerialEM.
SubareasBad	Set this entry to 1 for a Tietz camera if taking an image of a subarea makes the next image of the full camera area be bad.
BadColumns	Specify a list of bad columns that will be replaced in all processed images (gain normalized or dark subtracted).  List the columns in increasing order using DM coordinates numbered from 0.  This is needed if gain normalization is being done in SerialEM.
HotPixels	Specify a list of hot pixels if X ray removal from dark references is going to be used, to prevent these pixels from being treated as X rays.  Each pixel is specified by an X and a Y coordinate.  Multiple pixels can be entered on one line, and multiple lines can be entered.
HotPixelsAreImodCoords	Enter 0 or 1 if hot pixel coordinates are numbered from 0 (DM coordinates) or from 1 (IMOD/3dmod coordinates) respectively.
HotColumns	Specify a list of hot columns which should not be analyzed for X rays, in DM coordinates numbered from 0.  Include the bad columns if they are hot.
DarkXRayAbsoluteCriterion	The absolute number of counts by which a pixel must exceed the mean of a dark reference to be treated as an X ray.  These should be counts before division by 2, if any, for a 16-bit camera.
DarkXRaySDCriterion	The number of standard deviations above the mean that a pixel value must be to be treated as a dark reference X ray.
DarkXRayRequireBothCriteria	Enter 1 if a pixel must exceed both the absolute and the standard deviation criterion to be treated as a dark reference X ray, or 0 if exceeding either criterion will do.
MaximumXRayDiameter	The maximum diameter of an area that will be replaced as an X ray in a dark reference, in unbinned pixels.
ShowRemoveXRaysBox	Set to 1 to enable the appearance of the “Remove X-rays” checkbox in the camera setup dialog.  This option should be enabled only if the particular camera generates images where X-rays can ruin the tracking.
BeamBlankShutter	The shutter number of the beam blanking shutter (0 or 1; a Tecnai should be set up so that this is 1.)
OnlyOneShutter	Set this entry to 1 if there is only one shutter driven by this camera.
SetAlternateShutterNormallyClosed	1 to issue a command upon initialization to make sure that this camera is configured so that the beam shutter is normally closed and the film shutter is normally open.
BuiltInSettling	The intrinsic pre-exposure time in seconds when using the regular shutter for a Gatan camera; this seems to correspond to the clear time of the camera.
StartupDelay	Average time in seconds between issuing a command to take a picture, and the start of the actual exposure sequence (clearing the CCD for a Gatan camera, or starting the exposure for a Tietz camera).
MinimumDriftSettling	The smallest drift settling that SerialEM should try to produce.  For a Gatan camera. it corresponds to half of the range of variability in the time that an exposure starts after image acquisition is requested by SerialEM.  [For a Tietz camera, this should be set to 0 or a small number like 0.01].
ExtraBeamTime	The amount of time to allow after the expected end of the exposure before microscope actions can be taken and the beam can be blanked the beam to avoid beam exposure during the readout.  For a Gatan camera, this should equal MinimumDriftSettling.  For a Tietz camera, the sum of StartupDelay and this timing factor is used to control how soon microscope actions can be taken after readout should begin, but no beam blanking is needed.
ExtraOpenShutterTime	When the shutter is opened to provide a pre-exposure that exceeds the built-in settling (pre-exposure), the time specified by this entry is subtracted from the additional pre-exposure desired to keep the pre-exposure from exceeding the desired length.
ShutterDeadTime	The amount of exposure time lost when the beam shutter is operated; and entry for this is needed on JEOLs and can be obtained by running the routine in the Calibration menu.
Retractable	Set to 1 to indicate that the camera is retractable
InsertionDelay	Delay in seconds after inserting the camera.
RetractionDelay	Delay in seconds after retracting the camera.
GIF	Set to 1 to indicate that this is a GIF camera
Order	An arbitrary number to indicate the order of the camera in the beam path; cameras with lower order can block the beam from reaching cameras with higher order.
HasTVCamera	Set to 1 to indicate that this GIF has a TV camera.
InsertTVToUnblank	Set to 1 to indicate that the TV camera must be inserted to keep this camera from blanking an upper camera.  This setting is needed with a GIF unless you have some other means to keep the GIF from blanking when the upper camera is in use.
FilmToCameraMagnification	Magnification ratio between the film plane and the camera.  This is used to provide a fallback pixel size if pixel size is neither directly calibrated nor deducible from image shift calibrations.
PixelSizeInMicrons	Actual size of unbinned pixels in the CCD chip.
ExtraRotation	Number that needs to be added to the Tecnai-reported rotation angle and the global extra rotation angle too determine image rotations for this camera.  Only relevant if rotations are not calibrated directly or indirectly.
RotationAndPixel	This entry specifies a set of calibration values for a single magnification.  It should contain the following numbers on the line: 1) Magnification index 2) Change in image rotation from the next lower mag to this mag (in degrees, CCW positive), or 999 if this was not measured 3) The measured rotation from the X axis to the tilt axis (in degrees, CCW positive), i.e., the rotation angle solved for in Tiltalign plus 90 degrees; or 999 if this was not measured. 4) The pixel size in nanometers, or 0 if this was not measured.
EndCameraProperties	This entry indicates the end of the block of properties for a camera.

Obsolete camera options

CanDriveDMBeamShutter	Set this entry to 0 to indicate that the installed version of DigitalMicrograph cannot drive the alternate shutter via scripting, and to override the plugin’s decision based on DM version number.  This should be needed only if a 3.7.1 version below 3.7.1.6 is installed.
CanUseDMOpenShutter	Set this entry to 0 or 1 to indicate that extra pre-exposure (drift settling) can or cannot be achieved by the DigitalMicrograph scripting command to open the shutter.  This should not be needed.
CanDriveDMDriftSettling	Set this entry to 0 to indicate that the installed version of DigitalMicrograph cannot specify a DM drift settling via scripting, and to override the plugin’s decision based on DM version number.  This should be needed only if a 3.7.1 version below 3.7.1.6 is installed.
MinimumBlankedExposure	If the beam shutter cannot be controlled through scripting in DigitalMicrograph, SerialEM must request an exposure longer than the desired one using the regular shutter and unblank the beam for the desired exposure time during this longer exposure.  This entry specifies the smallest extended exposure time that will be requested.
ExtraUnblankTime	When SerialEM must produce an exposure by beam unblanking as just described, this entry is needed to specify how much time to subtract from the desired exposure time to make the actual unblanked time be correct on average.

File Options

These properties determine the default options that are selected when the file properties dialog box is opened.

FileOptionsMode	0 for bytes or 1 for 16-bit integers
FileOptionsExtraFlags	The sum of 1 to store tilt angles, 4 to store stage position * 25, 8 to store magnification / 100, and 16 to store intensity * 25000
FileOptionsMaxSections	Default value for the maximum number of sections, which determines the amount of space allocated for the extended header
FileOptionsPixelsTruncatedLo	Number of pixels that will be truncated at 0 when scaling integers to bytes
FileOptionsPixelsTruncatedHi	Number of pixels that will be truncated at 255 when scaling to bytes
FileOptionsUnsignedOption	Determines how 16-bit data will be stored as signed integers: 0 to truncate at 32767, 1 to divide by 2, 2 to subtract 32768
FileOptionsSignToUnsignOption	Determines how signed data are written to an unsigned integer file: 0 to truncate negative numbers at 0, or 1 to add 32768

Microscope properties

DeltaZtoDefocusFactor	Factor needed to adjust for a discrepancy between the change in defocus reported by the microscope and the actual defocus change.  Determined by calibrating autofocus, moving in Z and measuring defocus; the factor is (defocus change)/(change in Z height)
ScreenCurrentFactor	Factor needed to give a calibrated current in the screen meter
SmallScreenFactor	Additional factor needed to get the same current reading when switching between main screen and small current, with the beam filling the main screen
LensNormalizationDelay	Time delay after projector lens normalization, in milliseconds
LowestMModeMagIndex	Index of the lowest magnification in M mode, needed to prevent the task routines or filter control from switching into LM mode, and to indicate that image shift calibrations are not congruent across the LM-M boundary
NumberOfSpotSizes	Number of spot sizes on the microscope; default is 11.
IntensityToC2Factor	This entry is no longer needed; instead, the factor is measured by a calibration procedure and stored in the Calibrations file
TiltAxisOffset	Distance from the optical axis to the tilt axis, as indicated by the Refine Eucentricity routine.  To determine this value, run the routine at 3-5 times near (within 100 microns) of the center of the grid.
StageLimits	Limiting coordinates for stage movement in microns: minimum and maximum X coordinate, and minimum and maximum Y coordinate.  The default is +990 or -990 microns; a 0 may be entered to use the default.
StageMoveDelay	Delay time in milliseconds from moving the stage to taking a picture.
FloatingCurrentMeterSmoothed	0 for no smoothing or 1 for smoothing of current readings when the floating current meter is opened
InvertStageXAxis	Set to 1 to indicate that the stage coordinate system is left-handed when projected onto the screen.  The axes of this coordinate system are assessed by noting the direction that the image moves when stage coordinate is increased in X or Y; if the direction for X is 90 degrees counterclockwise from the direction for Y then this property is needed (and a global rotation of 180 degrees may also be needed.)
CurrentMeterLogBase	Base value added to screen current in nanoamps, before taking the log for smoothing.  Adjust this to make the smoothing behave similarly for large and small currents.
CurrentMeterSmootherThreshold1	Threshold change for a single value to trigger a change in the displayed, smoothed value (in log units).
CurrentMeterSmootherThreshold2	Threshold change in the running mean that will trigger a change in the displayed value (in log units).  Change these two thresholds together to adjust smoothing.
WatchGauge	Add a gauge to be watched for the VAC indicator in the microscope property panel.  Follow this entry with the official name of the gauge (e.g., column vacuum IGP1 is P4), the threshold between a green and yellow reading, and the threshold between a yellow and red reading, in Pascals.  A formula based on several simultaneous observations of Pascals and FEI log units for IGP is: Log reading = 26.2 * log(Pascal reading) + 135 Or, Pascals = 10 ** ((log reading – 135) /26.2)
MicronsPerUnitImageShift	A parameter that is used in the initial calibration of image shift (or when calibrating “from scratch”).  If image shift is not properly calibrated on the scope, this may need to be set to something besides 1.
MaxCalibrationImageShift	Maximum image shift change when calibrating image shift
MaxLMCalibrationImageShift	Maximum image shift change when calibrating image shift at LM-mode mags.
StageCalibrationBacklash	Microns of movement to adjust for backlash in the stage calibration procedure.  The default is 3 microns but 10 microns was needed on a JEOL.
MagnificationTable	A table of magnification index, film and screen magnifications, and the rotation angles reported by the Tecnai.  The line with this entry must contain the number of table entries to follow, then there must be that many lines following.  The form of each line is:      Index    Film mag    Rotation angle    Screen mag The first three entries can be obtained by selecting List Mags in the Calibration menu.  The screen mag must be added by hand.  If there is a GIF, each line should have three more entries:     Mag with screen up    Rotation angle    Mag with screen down
ImageShiftDelays	A table of delays to apply after an image shift.  The delays are applied before taking an image, and they are scaled up or down in some situations.  The line with this entry must contain the number of entries to follow, then there must be that many lines of the following form:     Distance in microns     Delay in seconds
LowDoseBeamNormDelay	In low dose mode, the condenser lenses can be normalized by passing through the intensity and spot size of the View area when changing areas (needed on the JEOL scope).  This value sets the delay in milliseconds after going to the View parameters (default 100 ms).

Autofocus and Autoalignment correlation properties

AlignTrimFraction	Fraction to trim off edges of images for autoalignment correlations
AlignTaperFraction	Fraction of image to taper to mean over for autoalignment correlations
AlignFilterSigma1	Sigma of inverted Gaussian that filters out low frequencies in autoalignment correlations
AlignFilterSigma2	Sigma fof Gaussian rolloff for filtering out high frequencies in autoalignment correlations
AlignFilterRadius2	Radius at which high-frequency Gaussian rolloff starts
FocusPadFraction	Fraction to pad images for autofocus correlations
FocusTaperFraction	Fraction of image over which to taper to mean for autofocus correlations
FocusFilterSigma1	Sigma of inverted Gaussian that filters out low frequencies in autofocus correlations
FocusFilterSigma2	Sigma of Gaussian rolloff for filtering out high frequencies in autofocus correlations
FocusFilterRadius2	Radius at which high-frequency Gaussian rolloff starts

Task operation properties

CheckAutofocusChange	The change in defocus that will be applied in the positive and negative direction when the routine to check autofocus is run.
ResetRealignMaxIterations	Maximum number of iterations when the routine to reset image shift and realign is run.
ResetRealignIterationCriterion	If image shift exceeds this criterion after resetting the image shift and realigning, another iteration will be done, up to the maximum number of iterations.
ResetRealignHigherMagCriterion	If image shift is less than this criterion when doing the reset and realign task, then the alignment pictures will be taken at one mag step higher than the default.
WalkUpMaxInterval	Maximum tilt interval for the walk up task.
WalkUpMinInterval	Minimum tilt interval for the walk up task.  Intervals will initially be the maximum interval times the cosine of the tilt angle, but will not become smaller than this minimum.
WalkUpShiftLimit	When image shift exceeds this limit during the walk up task, the reset and realign operation will be run.
WalkUpLowDoseISLimit	A similar limit on image shift, applied when walking up in low dose mode.
EucentricityBacklashZ	Amount to move in Z to correct for backlash when changing Z during the eucentricity routines.
EucentricityCoarseInitialAngle	Starting tilt angle for the coarse eucentricity task.
EucentricityCoarseInitialIncrement	Starting tilt increment for the coarse eucentricity task.
EucentricityResetISThreshold	Image shift will be reset to zero, and the stage moved to compensate, if it exceeds this criterion before a eucentricity task.
EucentricityCoarseMaxTilt	Maximum tilt angle for the coarse eucentricity task.
EucentricityCoarseMaxIncrement	Maximum tilt increment for the coarse eucentricity task.
EucentricityCoarseTargetShift	The coarse eucentricity routine will increase the tilt increment to try to make the image shift by this amount in microns.
EucentricityCoarseMaxIncrementChange	Maximum factor by which the tilt increment will change from one trial to the next in the coarse eucentricity task.
EucentricityFineIterationLimit	Maximum number of iterations for the fine eucentricity task.
EucentricityMaxFineIS	Maximum image shift for the fine eucentricity task before it changes Z height and starts a new iteration.
ResetRealignMinField	Minimum camera field of view, in microns, for resetting image shift and realigning, which determines the mag used.
ReverseTiltMinField	Minimum camera field of view, in microns, for the reverse tilt task.
EucentricityCoarseMinField	Minimum camera field of view, in microns, for the coarse eucentricity task.
EucentricityFineMinField	Minimum camera field of view, in microns, for the fine eucentricity task.
EucentricityFineAlignMinField	Minimum camera field of view, in microns, for doing fine eucentricity and maintaining image alignment.
WalkUpMinField	Minimum camera field of view, in microns, for the walk up task.
TiltAfterMoveMinField	Minimum camera field of view, in microns, for realigning after a tilt following a stage move.  If the value is non-zero, this task is invoked during walk up and tilt series on the tilt after an image shift reset.  The default is 0. for Tecnai and 4.5 um for JEOL.
TiltBacklash	Amount to tilt to eliminate tilt backlash.
MinTaskExposure	Minimum exposure time allowed, when it is necessary to reduce the exposure time upon going to a lower magnification (i.e., when intensity cannot be reduced enough).  This minimum will apply both for tasks and for low magnification tracking in a tilt series.  The default is 0.002, but a higher value may be needed if camera shuttering is slow and exposures of this duration give no image
RefineZLPStepSize	The step size in eV for the Refine ZLP procdure.  Default is 2.
RefineZLPSlitWidth	The slit width used for the Refine ZLP procedure.  Default is 20 eV.
RefineZLPMinimumExposure	The minimum exposure used in the refine ZLP procedure, in seconds.  The procedure uses the Trial parameter is normal mode and the Preview parameter set in Low Dose mode, with the exposure reduced by a factor of 10, but not below this minimum.  The default is 0.005, but numbers as low as 0.002 can be used if the number of counts is consistent within a few percent from one exposure to the next.
EnergyShiftCalMinField	Minimum camera field size for the lowest mag that will be done when calibrating mag-dependent energy shifts.  This should be set to the largest minimum field size for any of the tasks.  The default is 8 microns.

Tilt series controller properties

TSDefaultStartAngle	Default angle for starting and ending tilt series, when controller is not invoked at high tilt
TSMaxUsableAngleDiff	Maximum tilt angle difference between images that can be cross-correlated; this value is reduced by the cosine of the tilt angle in deciding whether an image is usable as a reference for alignment.
TSBadShotCrit	The fraction of expected counts that an image must contain to be accepted and not considered a bad shot.
TSBadLowMagCrit	The corresponding fraction for images obtained at a lower magnification, where intensities may not be set as accurately.
TSMaxTiltError	Maximum error allowed in setting tilt angle before the TSC decides that a pole touch has occurred.
TSLowMagFieldFrac	When low mag tracking is enabled, it will be used if the error in the last prediction was bigger than this fraction of the field.
TSStageMovedTolerance	When resuming from a stop, the user will be assumed to have moved the stage and disturbed predictability if any one of X, Y, or Z is bigger than this number.
TSUserFocusChangeTol	When resuming from a stop, focus predictability is considered disturbed if the difference between microscope defocus and the autofocus target has changed by more than this amount.
TSFitDropErrorRatio	When doing a fit, if the ratio of the standard error with all points included to the best error with the maximum number of points excluded is bigger than this value, points will be dropped.
TSFitDropBackoffRatio	Once the decision is made to drop points, points will be dropped until the standard error falls below the minimum standard error times this ratio.
TSMaxImageFailures	The maximum number of retries after a image is acquired that does not have enough counts.
TSMaxPositionFailures	The maximum number of retries after a Record image is acquired that loses more than the desired fraction of necessary image area.
TSMaxDisturbValidChange	Maximum number of disturbances to look past for valid changes to include in the fits.  A bad idea.
TSMaxDropAsShiftDisturbed	Maximum number of points to drop routinely from the fits for X or Y after a disturbance in position.
TSMaxDropAsFocusDisturbed	Maximum number of points to drop routinely from the fits for Z after a disturbance in focus.
TSXFitInterval	Maximum angular interval over which to fit X coordinates.
TSYFitInterval	Maximum angular interval over which to fit Y coordinates.
TSZFitInterval	Maximum angular interval over which to fit Z coordinates.
TSXMinForQuadratic	Minimum number of points to fit X coordinates to a quadratic curve rather than a line.
TSYMinForQuadratic	Minimum number of points to fit Y coordinates to a quadratic curve rather than a line.
TSMinFitXAfterDrop	Minimum number of points to retain in fit for X when dropping points.
TSMinFitYAfterDrop	Minimum number of points to retain in fit for Y when dropping points.
TSMinFitZAfterDrop	Minimum number of points to retain in fit for Z when dropping points.

Beam intensity calibration properties

Note that these properties changed completely with the change to CCD-based calibration in SerialEM 2.4.1

BeamCalMinExposure	Minimum exposure time for calibration images (default 0.1 sec)
BeamCalMaxExposure	Maximum exposure time for calibration images (default 3 sec)
BeamCalMinCounts	The minimum number of counts in a calibration image (unless exposure time is at the maximum), before division by 2 for 16-bit camera, if any.  The default is 100.
BeamCalMaxCounts	The maximum number of counts in a calibration image, before division by 2 for a 16-bit camera, if any.  This should be set to about 2/3 of your camera’s saturation level.  The default is 8000.
BeamCalSpacingFactor	Spacing between brightnesses measured, as a multiplicative factor (default 1.1)
BeamCalChangeDelay	Delay in milliseconds after setting a new beam intensity (default 400).
BeamCalInitialIncrement	Initial change in C2 intensity value (default 0.0002).
BeamCalMinField	The minimum camera field of view at which beam calibration will be needed; should correspond to the biggest minimum field size required for any of the tasks (default 8 microns)
BeamCalExtraRangeNeeded	Additional range in intensity to be calibrated beyond that needed to get the same intensity upon changing from the current mag to the lowest mag needed (default 10.)

JEOL properties

JeolUpdateByEvent	Set to 0 to disable updating by events if this screws up.
UpdateSpectroscopyByEvent	Set to 1 if the JEOL interface makes the promised event call when changing between spectroscopy and imaging modes.
InitializeJEOLDelay	The delay between initializing the JEOL COM object and the first attempt to call functions, in milliseconds, if events are not being used for update.  Without this delay, errors occur.
ScopeUpdateInterval	The interval in milliseconds between calls to the function that reads scope properties and updates the user interface.  The default is 100.  In the JEOL version, the update function does not normally access the microscope so this should be OK.
JeolUpdateSleep	The sleep time, in milliseconds, between calls to the update function that actually queries the scope in a separate thread.  If events are used for update, this function does one or two queries per call, taking up to 300 msec, so this interval must be long enough to leave substantial time for user interface handling, etc.  If events are not used for update, the stage position and status calls take up to 600 msec.
JEOLReportsSmallScreen	Set to 0 to indicate that the scope does not report the small screen position.  Needed on 2100.
JeolControlsBeamValve	Set to 0 to indicate that the gun valve cannot be controlled (non-FEG).
HasOmegaFilter	Set to 1 to indicate that the scope has an omega filter.  The program will then enable the filter control panel but not do any of the mag changes, etc, involved in EFTEM mode.
JeolForceMDSmode	Set to 1 to force the microscope into MDS mode so that image shift will not be reset when magnification is changed.
JeolPostMagChangeDelay	Some versions of TEMCON generate errors when stage parameters are accessed too soon after a magnification change.  This entry can take one or two delay times in milliseconds.  The first number specifies the delay for any scope accesses after the program changes mag.  The second number provides for an additional time during which access to the stage will be deferred.  If only one number is used, values of 2000 or more may be needed to prevent errors.  With two numbers, values of 1000 and 3000 could be tried.  Default is 0.
MagChangeFixISdelay	If updating by event, the maximum possible interval from the time when a neutral image shift is reported to the time when the mag change responsible for the neutralization is reported.
OtherShiftBoundaries	List of mag indexes at which image or beam shift calibrations are not congruent with the next lower mag.  Just like LowestMModeMagIndex, such an index indicates the start of a new mag range.  Within a mag range, both the relation between image shift output and position on the specimen, and the relation between beam shift output and position on the specimen, should be constant.
JEOLStageMotorRounding	The value to which stage coordinates will be rounded before being used to drive the stage motors, in microns.  Should be 0.001; with the emulator 0.1 is needed.
JEOLStagePiezoRounding	If this value is non-zero, then after stage coordinates are rounded as indicated by JEOLStageMotorRounding, the remainder will be rounded by this value (in microns) and used to drive the piezoelectric positioners.  With a value of zero, no piezo positioning will be done.  Keep this at 0.
JEOLObjectiveLensToMicrons	Scale factor for converting the objective lens fine digital readout to microns of defocus.  The default is 0.0058, the value for 300 KV; it may need to be changed to make the defocus values in SerialEM match the values on the microscope display.  For 200 KV the value should be 0.002.
JEOLObjectiveMiniToMicrons	Scale factor for converting objective minilens values to microns in low mag mode.  If no value is entered, objective lens values will be shown at all mags.
JEOLBeamShiftToMicrons	Scale factor for rough conversion of beam shift values to microns.  The default is 0.025, the value for 300 KV, which also works at 200 KV.  It needs to be changed to get big enough spot movements in the first round of calibrating beam shift.
JEOLLowMagBeamShiftToMicrons	Scale factor needed for calibrating beam shift in low mag mode.  If this is omitted, the factor specified in JEOLBeamShiftToMicrons is used.  A value of 0.15 works at 200 KV.
MicronsPerUnitImageShift	A parameter that is used in the initial calibration of image shift (or when calibrating “from scratch”).  The right value is needed to get shifts that are big enough, but not too big, in the first round of calibration.  A value of 0.02 is needed at 200 KV; a value of 0.1 is needed at 300 KV.
LowMagMicronsPerUnitIS	Scaling factor needed for initial calibration image shift in low mag mode.  If this omitted, the factor specified in MicronsPerUnitImageShift is used.  A value of 1 is needed at 200 KV, 1.5 at 300 KV.
JeolUseProjectorForIS	Set to 1 to substitute projector shift for image shift.  Neutral value, image shift, and beam shift calibrations would need to be redone and kept in a separate calibration file when operating in this mode, and different values of the microns per unit image shift values may be needed.
MessageBoxWhenClipIS	Set to 0 to avoid having a message box appear when image shift goes beyond range and gets clipped to its limiting values.


File menu commands

The File menu contains commands for opening, writing to, and reading from MRC image files, and for saving a Log Window:

Open New	Opens a new MRC file for saving images.
Open Old	Opens an existing image file.
Montage Setup	Starts dialog for setting montage parameters.
Close	Closes an open image file.
Save A	Saves image in Buffer A to file.
Save Active	Saves image in active window to file.
Overwrite	Saves image, overwriting a section in the file.
Save to Other	Saves image in active window to a file other than the open image file.
Set Truncation	Sets the amount of data that will be truncated if data are saved as bytes.
Set 16-bit Policy	Sets how 16-bit data will be treated when saved to file.
Set Signed Policy	Sets how signed data are treated when saved to unsigned mode file
Read	Reads an image from a file.
Read from Other	Reads an image from a file other than the open file.
Read Piece	Reads one piece from a montaged image file.
Open Log	Opens a log window to record program messages.
Save Log	Saves the log window to a file.
Save Log As	Saves the log window to a different file.
Read & Append	Reads from an existing log file and appends output to it.
Exit	Exits SerialEM.
Open New command (File menu)

Use this command to open a new MRC file for saving single-frame images.  You will first encounter the File Properties dialog, then the standard Save As dialog box.  If the file already exists, you will be asked to confirm that you want replace it.

You do not need to open a file before saving; if you choose the save command before a file is open you will go through the same sequences of steps to open a file.

You can open an existing image file for reading and writing with the Open Old command.  You can also read from a different file once one has been opened, using the Read from Other command.

Open Old command (File menu)

Use this command to open an existing image file for reading images from and saving images to.  You will enter the File Open dialog box  to select a file.  If the file is a montage, then montaging will be activated.

Shortcuts

Keys:	CTRL+O

Montage Setup command (File menu)

Use this command to open a file for montaging, or to check parameters after montaging has been started.  When starting montaging, you first enter the Montage Setup dialog box, which allows you to specify the number of frames, magnification, and binning.  From there, you enter the File Properties dialog box then the Save As dialog box.

Montaging can be started only if there is no current file open for conventional saving of single frames.  To start montaging, first close the open file if necessary.  To get back to saving single images after montaging, close the open montaged file.

Close command (File menu)

Use this command to close an open image file.  This will terminate montaging if the file is a montage.

Save A command (File menu)

Use this command to save the image in Buffer A to the open image file.  If there is no image file open yet, you will first enter the File Properties and Save As dialog boxes to specify a file.  This file will become the open image file, available for further saving.  However, only images of the same size can be saved in the same file.

Shortcuts

Keys:	CTRL+S

Save Active command (File menu)

Use this command to save the image in the active buffer to the open image file.  If the main window is active, then the buffer currently displayed in the main window will be saved.  If  some other window is active (has the input focus, with its title bar highlighted rather than gray), then its image will be saved.

If there is no image file open yet, you will first enter the File Properties and Save As dialog boxes to specify a file.  This file will become the open image file, available for further saving.

Overwrite command (File menu)

Use this command to overwrite an existing image in the open image file with the image in Buffer  A.  You will be prompted to enter the number of the section that you wish to overwrite; the default value offered to you is the number of the last section in the file.  Sections are numbered from 0.

If you are montaging and need to overwrite a section, you will need to change the current Z value in the Montage Control Panel.

Save to Other command (File menu)

Use this command to save the active image into a file other than the open image file.  Only a single image can be saved into such a file using this command.  You will enter the File Properties and Save As dialog boxes, as usual.  If you specify an existing file, it will be replaced.  The file will be left closed when you are done.

Set Truncation command (File menu)

Use this command to modify the truncation of the data when saving images as bytes.  When one chooses to save bytes, then SerialEM can avoid compressing the dynamic range of the data by truncating the values of a relatively few pixels.  The number of pixels to truncate at the black and white end of the range is initially specified in the File Properties dialog, but can be changed thereafter with this command.

Set 16-bit Policy command (File menu)

Use this command to modify the way that data from a 16-bit will be treated when they are saved as integers in a signed data file.  You will be asked to enter 0 to truncate the data at 32,767, which could give saturated images; 1 to divide the data by 2, which will discard probably useless precision; or 2 to subtract 32768, which will preserve the data in its entirety at the expense of having negative numbers.  If a file is reopened, this is the way to set the treatment of the data to be different from the default.

Set Signed Policy command (File menu)

Use this command to modify the way that signed data are saved to a file that is in unsigned 16-bit integer mode.  By default positive values are saved without modification, and negative values are truncated at zero.  Enter 1 to have 32768 added before values are saved, which will preserve negative values.

Read command (File menu)

Use this command to read an image from a file.  If there is a currently open file, the image will be read from it; otherwise, you will enter the File Open dialog box to select a file.  In that case, the file will not remain open after reading from it.

Unless there is only a single section in the file, you will be asked to enter the section number to read; the default value offered to you is the number of the last section in the file.  Sections are numbered from 0.

If you are reading from a regular file with single frames, the image will be read into the buffer selected in the Buffer Control Panel.  If the file that you are reading from is a montage, then SerialEM will read the entire section and leave a binned-down overview in Buffer B and a full-scale image of the center of the area in Buffer A.

Read from Other command (File menu)

Use this command to read an image from a file other than the currently open image file.  You will enter the File Open dialog box to select a file.  Then, unless there is only a single section in the file, you will be asked to enter the section number to read; the default value offered to you is the number of the last section in the file.  Sections are numbered from 0.

The image will be read into the buffer selected in the Buffer Control Panel unless the file is a montage, in which case SerialEM will read the entire section and leave a binned-down overview in Buffer B and a full-scale image of the center of the area in Buffer A.  It is possible to read from a montaged file even if montaging is currently activated with a different file open.

Read Piece command (File menu)

Use this command to read in a single frame (piece) from an open montaged image file.  Unless there is only a single section in the file, you will be asked to enter the section number to read; the default value offered to you is the number of the last section in the file.  Sections are numbered from zero.

After specifying the section, you will be asked to enter the number of the piece in X and Y; pieces are numbered from 1.  The image will be read into the buffer selected in the Buffer Control Panel.

Open Log command (File menu)

Use this command to open a Log Window for recording messages from the program.  Once the window is open, some messages will be logged there instead of presented in message boxes.  You can control how much output goes to the window by selecting Verbose in various menus (Focus, Tasks, Tilt Series).

Save Log command (File menu)

Use this command to save the contents of the Log Window to a file.  You will enter the Save As dialog box to specify the file, which will have the default extension .log.  Once the window has been saved, selecting this command will save it again to the same file as the last time that it was saved.

Save Log As command (File menu)

Use this command to save the contents of the Log Window to a different file from one already specified.  You will enter the Save As dialog box to specify the file, which will have the default extension .log.  Once you save to a file with this command, using the Save Log command will save to this file.

Read & Append command (File menu)

Use this command to append the contents of the Log Window to an existing log file.  You will enter the File Open dialog box to select a file.  The program will then read in the contents of this file, place it before any text already in the Log Window, then write the window back out to the file.

Exit command (File menu)

Use this command to end your SerialEM session.  You can also use the Close command on the application Control menu.  SerialEM prompts you to save a log window with unsaved changes or to save settings if you do not have an open settings file.

Shortcuts

Mouse:	Double-click the application's Control menu button.

Keys:	ALT+F4


Settings menu commands

The Settings menu offers the following commands:

Open	Opens and reads a settings file.
Read Again	Rereads settings from the current file.
Save	Saves settings to file.
Save As	Saves settings to a new file.
Close	Closes settings file to prevent saving to it upon exit
Read Defaults	Reads system default settings file.
Autosave	Periodically save settings to currently open file.

About Settings Files

Settings files are used to store just about every parameter that can be set by you in SerialEM.  The main exceptions are program states such as Montaging, Low Dose mode, and Tilt Series mode, which need to be activated by you each time you run the program.  By default, SerialEM will remember all of your parameters from one session to the next in a file called SerialEMsettings.txt in the directory where SerialEM was started.  One can use the options in this menu to save settings into other files and retrieve settings from such files.

After parameters are read from a file, that file is left open, which has three implications: 1) Settings can be saved to that file via the entry in this menu; 2) Settings can be restored from the file to the value that they had when they were last saved there; and 3) When you quit the program, settings will be saved into that file.  To prevent settings from being saved into a file when you quit the program, just Close the file.

If your settings become too far from usable, you can restore them to default values by reading from a file called the System Default Settings file.

Macros are also stored in settings files.  When you read a file that contains macros, the macros in the file will replace the respective ones in the program, but any other macros that you have defined will be retained.

Open command (Settings menu)

Use this command to open a file in which settings were stored previously.  Ordinarily, your default settings file is open whenever you are running SerialEM.  Thus, opening a new file will usually involve closing that file.  If this is the case, you will be asked whether you want to save your current settings into the currently open file before closing it and opening a different one.

You will enter the File Open dialog box to select the file, which is a text file.

Read Again command (Settings menu)

Use this command to read parameter values from the currently open settings file.  You would do this if you do not want to keep any parameter changes that you have made and want to restore your settings to the state saved in this file.

Save command (Settings menu)

Use this command to save settings to a file.  If there is a settings file open, then the program state will be saved to this file.  If not, you will enter the Save As dialog box to specify a new file to save values into.  A file that has been saved into is left open; in other words, it will be saved to again when the program exits, unless you close it first.

Save As command (Settings menu)

Use this command to save settings to a file different from the one that is currently open.  You will enter the Save As dialog box to specify a file to save values into.  Any previously open settings file will be closed, and the new one will be left open.  This means that it will be saved to again when the program exits, unless you close it first.

Close command (Settings menu)

Use this command to close a settings file.  This means that it will not be saved to when the program exits, which you might want to do if you are going to experiment with parameters that you will not want to save.  You will be asked whether you want to save your current settings to the file before closing it.

Read Defaults command (Settings menu)

Use this command to read the System Defaults settings file.  If there is a settings file currently open, you will be asked whether you want to save your settings to that file before overlaying them with the system defaults.

Autosave command (Settings menu)

Use this command to control whether settings will be saved to a file periodically (every 5 minutes, except during a tilt series).  Autosaving is useful if you are doing things that present a risk of crashing the program.  If no settings file is open when Autosave is on, then nothing will be saved.


Camera menu commands

The Camera menu offers the following commands:

Parameters	Opens dialog box to set parameters for image acquisition.
Prepare Gain Ref	Opens dialog box to acquire a gain reference in SerialEM.
View	Acquires image with first parameter set.
Focus	Acquires image with second parameter set.
Trial	Acquires image with third parameter set.
Record	Acquires image with fourth parameter set.
Preview	Acquires image with fifth parameter set.
Montage	Starts montage acquisition to file.
Prescan	Acquires binned-down montage without saving.
Stop Acquire	Stops image acquisition.
Show Gain Ref	Shows the gain reference last used to normalize an image.
Show Dark Ref	Shows the dark reference last used.
Post-actions	Allows actions like tilt and image shift right after exposure.
Divide 16-bit by 2	Divides data from 16-bit camera by 2 in camera plugin
Normalize Here	Toggles gain normalization within SerialEM on and off.
Set Timing	Set timing parameters for dual-shuttering mode.
Set Corrections	Set basic image corrections performed in DigitalMicrograph
Screen Down	Leaves screen down during image acquisition.
Simulation	Gets simulated images rather than acquiring from camera.
Debug Mode	Outputs debugging information to Log Window and Digital Micrograph Results window

Parameters command (Camera menu)

Use this command to open the Camera Setup dialog box and set parameters like exposure time, field of view and binning for the 5 different exposure modes.

Shortcut Key:	CTRL+P

Prepare Gain Ref command (Camera menu)

Use this command to open the Gain Reference dialog box and acquire a gain reference that will be used for gain normalization within SerialEM.

View command (Camera menu)

This command acquires an image with the first camera parameter set.  This would be a good parameter set to use for highly binned, continuous exposures, if continuous exposures worked reasonably well.

In Low Dose mode, images captured with these parameters are not shifted away from the center of the recording area.  The View area is intended to be set up to give a low magnification, low exposure overview.  The various procedures which would ordinarily lower the magnification to track positions, such as the eucentricity-finding routines, use View exposures in low dose mode.  In other words, these routines rely on the parameter set giving adequate exposures for low-mag tracking in low dose mode.

Shortcut Key:	CTRL+V

Focus command (Camera menu)

This command acquires an image with the second camera parameter set.  This parameter set is used by the Autofocus routine to acquire two or three images in quick succession, so it is typical to set it up for a small image that can be read out quickly, such as the central quarter of the field, with somewhat short exposure and drift settling times.  It would be nice if one could also use this parameter set for visually assessing focus, but one usually wants a larger area for that purpose.

In Low Dose mode, images captured with these parameters can be, and typically are, shifted away from the center of the recording area.

Shortcut Key:	CTRL+F

Trial command (Camera menu)

This command acquires an image with the third camera parameter set.  This parameter set is typically set up to acquire a full-frame image relatively quickly.  It is used extensively by various routines that do tracking (e.g., eucentricity-finding routines).  You should thus make sure that this parameter set gives an adequate exposure for tracking.  Many of these routines actually compose a separate parameter set, changing the binning and exposure time if necessary so that binned images of no more than 512 pixels are acquired.  However, when the tilt series controller acquires tracking images, it uses the Trial parameters without modification.  It is recommended that you set these parameters to bin images binned down to no larger than 512 pixels, and that you acquire a full-frame image unless you have some specific reason not to.

In Low Dose mode, images captured with these parameters can be, and typically are, shifted away from the center of the recording area.

Shortcut Key:	CTRL+T

Record command (Camera menu)

This command acquires an image with the fourth camera parameter set.  This parameter set is intended to be used for final acquisition of images to be saved, and the Tilt Series Controller will rely on it being set up for that purpose.  In addition, montaging is done by acquiring images with these parameters.

In Low Dose mode, images taken with these parameters define the center of the recording area and can be considered unshifted.

Shortcut Key:	CTRL+R

Preview command (Camera menu)

This command acquires an image with the fifth camera parameter set.  This is probably useful only in Low Dose mode, where it acquires an image from the Record area, using the same magnification and beam settings as for Record images.  It can be set up to provide a very low exposure image of the Record area.

Shortcut Key:	CTRL+L

Montage command (Camera menu)

This command will start the acquisition of a montaged image, with the pieces saved to file.  If montaging has not been started yet, you will first enter the Montage Setup dialog box, which allows you to specify the number of frames, magnification, and binning.  From there, you enter the File Properties dialog box then the Save As dialog box.

Shortcut Key:	CTRL+M

Prescan command (Camera menu)

This command will acquire a binned-down montaged image, which allows a quicker assessment of the area that will be captured in an actual montage.  The images are not saved to file.  However, montaging needs to be set up first, which does entail defining an output file.  If montaging has not been started yet, you will first enter the Montage Setup dialog box, then ensuing dialogs for opening a file.

Stop Acquire command (Camera menu)

This command will stop image acquisition if it is still in some intermediate state, such as raising the screen or acquiring a dark reference.  However, once the request for an image has been issued to Digital Micrograph, stopping will have no effect.

Show Gain Ref command (Camera menu)

This command will place the gain reference that was last used to normalize an image into buffer A and roll the contents of A into B, etc., to the extent that buffer rolling is selected.  The gain reference consists of real numbers around 1.0 that are multiplied by the image to normalize it.

Show Dark Ref command (Camera menu)

This command will place the last-used dark reference into buffer A and roll the contents of A into B, etc., to the extent that buffer rolling is selected.

Post-actions command (Camera menu)

This command toggles a mode in which various time-consuming actions are initiated immediately after camera exposure ends, at the beginning of the image readout.  These actions can be magnification changes, image shift, and stage tilting.  Stage tilting is the most noticeable action, because it means that when you stop the Tilt Series Controller, it will often have already gone on to the next tilt.  If this behavior is problematic, you can disable it with this command.

Divide 16-bit by 2 command (Camera menu)

This command toggles a mode in which all images from a 16-bit camera are divided by 2 in the camera plugin, prior to being received by SerialEM.  In this mode, negative numbers in a dark-subtracted or gain-normalized image will be preserved instead of truncated at zero.  This could be important for low-exposure images if the dark reference drifts downward.  Another advantage of this mode is that the numbers shown in SerialEM will match those saved to a file.  When this mode is selected, the options for treatment of 16-bit data in the File Properties dialog box will be disabled because they are no longer relevant.  (The image has already been divided by 2 and will not be recognized as an unsigned 16-bit image.)

Normalize Here command (Camera menu)

This command toggles the processing (dark subtraction and gain normalization) of images within SerialEM.  This setting is initially determined by the ‘GainNormalizeInSerialEM’ property.  When it is off, processed images are acquired from DigitalMicrograph.  When it is on, raw images and dark references are acquired from DigitalMicrograph, and the processing is done in SerialEM.  In the latter case, the gain reference could come from DigitalMicrograph or from one prepared in SerialEM, depending on which exists.

Set Timing command (Camera menu)

This command allows you to enter values for the startup delay, minimum drift settling, and extra beam time needed in the dual shuttering mode of operation.  The option is enabled by being in Administrator mode.

Set Corrections command (Camera menu)

This command allows you to enter a value for the basic corrections to be done when DigitalMicrograph acquires an image.  This value may initially be set with the ‘BasicCorrections’ property, otherwise it is -1, meaning that default corrections will be done.  The value should be the sum of 1 for defect correction, 16 for bias correction, and 32 for linearization.  0 will turn off all default corrections.  This command is enabled by being in Administrator mode.

Screen Down command (Camera menu)

This command is used to toggle a mode in which the screen is not raised before a camera exposure.  This could be useful for looking at the beam during an exposure.  It is also needed to get images in simulation mode when there is no Tecnai microscope (TemServer) running.

Simulation command (Camera menu)

This command is used to toggle simulation mode, in which fake images are produced within SerialEM rather than acquired from DigitalMicrograph.  It should not be needed for simulation if DigitalMicrograph is started with Ctrl and Shift held down to activate the Faux camera.

Debug Mode command (Camera menu)

This command is used to toggle debug mode, in which the SerialEMCCD plugin will chronicle its transactions in the DigitalMicrograph Results window.  The camera controller in SerialEM will also output some timing information in the Log Window.


Calibration menu commands

The Calibration menu offers the following commands:

Image Shift	Calibrates image shift at the current magnification.
IS from Scratch	Calibrates image shift, ignoring any previous calibration.
Stage Shift	Calibrates stage shift for the current magnification.
List IS Vectors	List image shift calibration vector lengths and angles
List Stage Cals	List stage calibrations converted to specimen to stage matrices
Beam Intensity	Calibrates beam intensity at current magnification and below.
Spot Intensity	Calibrates relative intensity of the different spot sizes
Beam Shift	Calibrates beam shift by taking pictures of a small spot.
Electron Dose	Calibrates electron dose for the current spot size with an existing image.
Purge Old Calibs	Removes dose calibrations prior to current run of the program
Autofocus	Calibrates autofocusing
Set Focus Range	Set defocus range and number of steps for focus calibration.
Mag Energy Shifts	Calibrates the energy shift of the zero-loss peak with change in magnification.
Distortion	Acquires overlapping pair of images for measuring distortion field.
Set Overlaps	Set the minimum and maximum overlap factors for acquiring distortion pairs.
List Mags	Outputs list of all magnifications and image rotations to the Log Window.
Set C2 Factor	Set scaling factors for C2 by entering the C2 readout for all spot sizes.
Beam Crossover	Set intensity at which beam crossover occurs for all spot sizes.
Neutral IS Values	Measures neutral image shift values at all magnifications (JEOL)
Camera Timing	Determines timing parameters for dual shuttering mode and post-exposure actions
Shutter Dead Time	Determines the minimum exposure time possible
Administrator	Enters Administrator mode for saving calibrations and getting more reports
Save Calibrations	Saves calibrations to system calibration file.
Set Debug Output	Allows debug output string to be entered after program startup

Image Shift command (Calibration menu)

This command starts calibration of image shift at the current magnification.  You should always calibrate image shift at the appropriate magnification when montaging.  Image shift is calibrated by taking 9 pictures at different shifted positions.  Every other picture is taken in the unshifted position, so that drift can be eliminated.  Exposures are done with images binned to 512 pixels, using an exposure time based on the settings in the Trial parameter set.

If image shift has already been calibrated at any magnification in the same magnification range, the procedure takes advantage of this information to set the image shifts of the shifted pictures.  Otherwise, calibration is done in two rounds, first with very limited shifts, then with more wide-ranging ones.  There are two magnification ranges for image shift calibrations: LM mode mags and all higher mags.

At the end, you will see a message about the calibration matrix that was measured, the amount of error in those values, the amount of drift that was detected during the sequence of pictures, and a statement about the quality of the calibration.  At this point, you may be offered the opportunity to change all other existing image shift calibrations in the same mag range by the same amount.  If your calibration is good, this will eliminate the need to recalibrate at the other magnifications.

The program will apply a shift to the pictures taken with image shift to show how they align with the pictures in the central position.  If you set up to roll buffers A through M in the Buffer Control panel before starting the calibration, then you can scroll through all the images and view the alignments.  This could be important for verifying the alignment when a calibration grating is used at relatively low magnification.

This procedure may fail if existing calibrations are far off, which can happen when the microscope alignment has been changed.  If this happens, you should run the IS from Scratch command .

IS from Scratch command (Calibration menu)

This command will calibrate image shift in two rounds (13 pictures), without relying on any existing calibrations.  This procedure is needed for the first calibration in a magnification range and may be needed when the image shift calibration in the microscope alignment has been changed by a large amount.

Stage Shift command (Calibration menu)

This command will calibrate the relation between stage movements and position on the camera at a particular magnification.  This is a difficult relationship to calibrate accurately, so the program will average all of the stage calibrations available to it when converting, for example, an image shift to a stage move.  This calibration is helpful if montages are going to be done with stage shift, such as when making maps with the Navigator.  It is recommended that you do this calibration at a magnification of 1000-2000x and at 3 or 4 lower magnifications.

Before doing the calibration, you should have measured and entered into the properties file at least one pixel size in the LM range and in the regular mag range, and the rotation angle between the lowest M-mode mag and the highest mag in LM mode.  You should also calibrate image shift for whatever mags you are going to do this calibration at.

You should have a specimen other than the replica grating for this calibration, and either one on a slot grid or one with relatively large spacing between grid bars.  Position the specimen so that there are image features useful for correlation throughout the camera field of view.  Start the procedure.  The program will take 6 pictures, first three overlapping pictures with movements of the stage in X then three with movements in Y.  At the end it will report the transformation matrix, the maximum variation between the two estimates of each factor, and the implied transformation from stage to specimen coordinates (provided that there is sufficient information to derive the latter).  The first and third images in each triplet will also be shifted to show how then align with the middle image.  Scroll through these images to make sure that A, B and C are aligned, and that D, E and F are aligned.  If there is a misalignment, if the variation between estimates is bigger than 10%, or if the stage to specimen transformation is not close to -1.0 0. 0. -1.0, you should redo the calibration.

List IS Vectors command (Calibration menu)

Use this command to get a listing of the length and angles of the image shift calibration vectors for all magnifications where image shift has been calibrated.  This listing can provide a check on the accuracy of the image shift calibrations and, on a JEOL, reveal magnification boundaries where there are discontinuities in the image shift calibration.  A length and an angle are computed for a change in the X image shift and for a change in the Y image shift.  The vector lengths represent the estimated microns moved on the specimen per nominal micron of applied image shift, so the lengths should be near 1.  The angles are angles on the specimen and they should stay constant as long as there is no discontinuity in image shift behavior.

These estimates are based on the pixel size and image rotation angle at each magnification, which can be obtained in one of two ways.  The pixel size can be derived from the nominal film magnification, the camera pixel size, and the magnification ratio from the film plane to the camera; while the image rotation angle can be derived from the magnification table.  Alternatively, a calibrated pixel size from the property file can be used whenever available, as well as a calibrated rotation angle.  (Note that if there is one mag with an absolute calibration of the angle and a contiguous series of mags with relative rotations entered, all of those mags are considered to have calibrated rotation angles).  The latter option will give the most accurate estimate of the vector lengths but one can expect to see small discontinuities in the lengths between the mags with and without calibrated pixel sizes.  Similarly, the angles will be most accurate but there will be discontinuities between mags with and without calibrated angles.  Thus, if you have calibrated only one or two pixel sizes, just use the estimates based on the nominal magnification (answer NO to the query).  If you have calibrated many pixel sizes, as is necessary on a JEOL, answer YES to use these pixel sizes and get the best indication of whether there are discontinuities that need to be specified with the OtherShiftBoundaries property.

List Stage Cals command (Calibration menu)

This command will convert the stage calibration for each magnification into a specimen to stage transformation and also show the average transformation that is being used regardless of mag.  This allows the consistency of the calibrations to be assessed at any time.  Transformations should be near -1. 0. 0. -1.

Beam Intensity command (Calibration menu)

This command will calibrate beam intensity for the current spot size, starting at the current intensity and going down to intensities needed at magnifications below 10000x.  SerialEM uses these calibrations to change intensity by a desired amount rather than to achieve an absolute intensity level.  The range of magnifications is needed so that the program can use the calibrations to set intensity properly when going to low magnification during various procedures.  This is preferable to simply relying on the intensity zoom feature.

As of version 2.4.1, the program uses the CCD camera instead of measuring screen current, giving more reliable calibrations with less difficulty.

To prepare a calibration, start with the brightest beam that anyone might need calibrations for.  In other words, go to the highest magnification that might be used for tilt series, and condense the beam until it is slightly bigger than the field of the CCD camera.  For an extra margin of safety, you could start one or two magnification steps higher.  Also, if the beam is too bright when condensed that much, you can start at a higher magnification and with the beam spread more.  Select this command to start the procedure, then check that the beam is still centered.  (On the Tecnai, the C1 lens (spot size) is normalized, which can affect the beam size and position).  The program will determine an appropriate binning for short exposures, then take images with successively lower intensity, increasing the binning, decreasing the magnification, and increasing the exposure time as needed to cover a very wide range of intensity.  Do this procedure without a specimen in place.

In Administrator Mode, the program will output the calibration table for each magnification, consisting of the number of unbinned camera counts per second at the starting magnification, and a C2 setting.  The third number on each line is the number of retries before getting a current in the right range at each step.  If images appear saturated, you may need to adjust the maximum number of counts, or the minimum exposure time.  For information on these and other properties controlling the procedure, see Beam intensity calibration properties

Spot Intensity command (Calibration menu)

This command will measure the relative intensity of the different spot sizes.  With this information, the program can take a dose calibration done at one spot size and use it to compute the dose at another spot size.  The procedure will take pictures with the Trial parameter set, starting at the brightest spot and working down to the dimmest spot without changing the other condenser lens.  Before you start it, you need to spread the beam to about the size of the screen, then go to Spot 1 and set the Trial exposure so that it gives counts about ½ to 2/3 of saturation.  Then step through the spot sizes and make sure the beam stays on the field of the camera – if not, spread the beam some more.  When you select this command on the Tecnai, the first condenser lens (spot size) will be normalized, so you should then check the centering and size of the beam before confirming that the procedure can be started.

Beam Shift command (Calibration menu)

This command will calibrate beam shift so that the beam can be moved by predictable amounts.

To do this calibration, go to a magnification near 20K.  First adjust the spot size and intensity so that the beam is a small, centered spot whose diameter is less than half the width of the CCD field (for a 2K, 30-micron pixel camera, this is 2-3 times the diameter of the smallest circle on the screen).  Take a Trial exposure to verify this, and adjust exposure time or spot size to prevent CCD saturation.  Then select this command to perform the calibration.  Seven pictures will be taken: one at the starting position, two with small movements of the X and Y beam shifts, and four with larger movements in both directions from center.

Electron Dose command (Calibration menu)

Use this command to calibrate electron dose for a particular spot size.  This calibration is automatically saved in a special short-term calibration file and is only good for a limited period of time (1 day).  The calibration requires that beam intensity be calibrated and that the SerialEMproperties.txt file contain an entry for CountsPerElectron for the CCD camera.  Before activating the command, take an image of the blank beam with a reliable exposure time (e.g., 0.2 seconds).  Select the command and press OK.  The program will report the dose rate (electrons per square Angstroms per second) of the current beam intensity.

The dose calibration will be most accurate if the spot size is normalized (on the Tecnai).  To do this, open the Normalizations panel in the Tecnai User Interface, select TEM Spotsize, and check Spotsize.  Then change spot size to get a normalized beam, take a picture, and calibrate the dose with it.  By the same token, the actual measurement of dose at a particular beam setting will be more accurate if the spot size is normalized whenever you change spot sizes.  Hysteresis in the second condenser lens will also reduce the accuracy of a dose measurement, but this is more difficult to control for.

The dose calibration is specific to the side of beam crossover on which the calibration image was taken.  Once a calibration is present, the program can compute the dose for any image acquisition when beam intensity is within the calibrated range, taking into account drift settling time as well as CCD exposure time.  If relative spot intensities have been calibrated, then a calibration at a single spot size will allow dose to be computed at any spot size.  Note that dose is automatically calibrated when you take a gain reference, unless you deselect this option in the Gain Reference dialog .

When there is an electron dose calibration, dose will be displayed in several places:

1)	The dose for a low dose acquisition area will appear in the upper right of the Low Dose control panel, with a continuous readout if “Continuous update” is selected.

2)	The dose for the current camera exposure time, in the lower right of the Camera Setup dialog .

Purge Old Calibs command (Calibration menu)

This command will remove any dose calibrations done before the current instance of the program was started.  You would use this if you do a dose calibration (or take a gain reference) and then want to remove any earlier calibrations.  Dose calibrations older than 24 hours are ignored when the program starts.  Also, when dose is calibrated, other calibrations older than 12 hours are then removed.

Autofocus command (Calibration menu)

This command will calibrate autofocusing at the current magnification.  This involves going to a series of defocus levels and measuring the amount and direction of image displacement between two pictures with the beam tilted by positive and negative angles.  Because this displacement does not vary linearly with the defocus, the calibration consists of a whole curve of displacements as a function of defocus, rather than just the slope and magnitude of a vector.  Nevertheless, the curve can be characterized for some purposes by a slope and magnitude, and these parameters are used to assess how much the curve has changed from the stored calibration.

The defocus levels sampled in the calibration are controlled by the Set Focus Range command .

Before calibrating autofocus, you should be sure that beam tilt pivot points are well-aligned, that there is no condenser or objective astigmatism, and that the Focus parameter set takes good pictures.  Calibrate with a specimen that does not require much drift settling, and do not calibrate at high tilt.  Use as high a beam tilt as is practicable to get the most accurate results (beam tilt can be set under the Focus menu).  The beam tilt during autofocusing does not need to match the beam tilt used for calibration.

Note that if image shift is calibrated at the various magnifications of interest, there is no need to have an autofocus calibration at more than one magnification.  It is better to have one high quality focus calibration over a large focus range rather than poorer calibrations at several magnifications.

Set Focus Range command (Calibration menu)

This command allows you to set the range and number of defocus levels that will be assessed when calibrating autofocus.  After you select the command, you will make entries to three dialog boxes:

1)	Total defocus range to measure shifts over: The program will sample defocus levels from half this range above the current defocus to half this range below the current defocus.

2)	Number of focus levels to test: This determines the interval of sampling; i.e., the total defocus range divided by this number minus 1.

3)	Number of levels to smooth over (0 for none): The entry controls the amount of linear smoothing of the curve, with no smoothing if 0 is entered.  Smoothing will reduce the effects of variability in the individual measurements.  A value of 3 is recommended.

Mag Energy Shifts command (Calibration menu)

Use this command to calibrate how much the zero loss peak shifts in energy when changing magnification.  This calibration involves taking many pictures with the Trial parameter set.  This procedure should be run with a uniform beam, i.e., no specimen.  For the calibration, do the following:

1)	Adjust the Trial parameters to give a quick picture with a moderate number of counts; for example, center ½ area, binned to 256x256, with an exposure that gives 4000 counts for a 15-bit 2K camera.

2)	Align the Zero loss peak at a mid-range magnification.

3)	Switch to Administrator mode to run the procedure from low to high then high to low mag, for the most accurate results.

4)	Start the calibration and respond to the series of dialog boxes.  First specify the highest mag that should be calibrated.  The default is to do 13 mags starting with the lowest mag in M mode.

5)	Specify the total energy range that will be scanned for the location of the zero loss peak.  The default should work unless you have big energy shifts.

6)	Specify the step in energy for scanning the energy range, and the slit width.  The defaults should work.

At each mag, the program will scan through the range of energies and measure the intensity of the image, finding the center of the energy range that lets the beam through unimpaired.  You should see images with very low counts at least at the beginning of each scan (the program will stop a scan when it decides that it has seen the peak).  If you do not, you may need to redo the procedure with a bigger scan range.

Remember to save your calibrations.

Distortion command (Calibration menu)

This command will allow you to capture a pair of images that can be used for measuring the microscope distortion field.  The pairs can overlap side by side, top to bottom, or diagonally.  The direction of overlap is specified by a number from 0 to 7.  Directions 4 to 7 are equivalent to directions 0 to 3, so you just need to use directions 0 to 3 to obtain a complete set of the different kinds of overlapping pictures that will give twice as much data as unknowns when solving for a distortion field.

Before starting, center the specimen on an area that has rich image information throughout.  Set up Record parameters for fairly high counts in images binned to 1Kx1K.  Select the command and specify a direction for the pairs.  The program will offer to set image shift to zero if you have not done so already.  Then it will proceed to take overlapping image pairs with stage movement between the two images of a pair, until it acquires a pair with overlap within acceptable limits.  It will try ten times.  If it fails, just try again, perhaps moving to a new area.

Set Overlaps command (Calibration menu)

This command allows you to set the limits within which overlap factors must fall for the distortion pairs.  You might need to expand the range of acceptable overlaps if you are trying to get pairs at a high magnification.  Images arranged side by side or one on top of the other must meet three criteria.  The overlap factor in the direction separating the images must fall between a minimum and a maximum value, and the overlap in the perpendicular direction must exceed a high minimum value.  There are two separate criteria for images arranged diagonally: the minimum and maximum overlap factors, which must be met separately in X and Y.  If you need to change these factors, try to expand the maxima more than you reduce the minima.

List Mags command (Calibration menu)

This command will cycle through all of the available microscope magnifications and print in the Log Window the magnification index, the film magnification, and the image rotation as reported by the Tecnai.  These numbers are listed in the form required in the SerialEMProperties.txt file.  Different numbers will be output in EFTEM mode than in normal lens mode.

Set C2 Factors command (Calibration menu)

Use this command to calibrate the factors that SerialEM needs to convert from the beam intensity values available to it into values matching the C2 lens percentage reported in the Tecnai User Interface.  Without this calibration, C2 values shown in the Low Dose control panel and in the electron dose calibration procedure will not match values shown by the Tecnai.  First be sure that you have a C2 lens readout in your TUI workspace.  Then activate the command and type in the value shown in the C2 readout for each spot size.

Beam Crossover command (Calibration menu)

This command allows a calibration of the C2 setting for beam crossover at each spot size.  This calibration is needed in order to maintain beam intensity calibrations on both sides of crossover.  After you select the command, the program will cycle though the spot sizes and ask you to bring the beam to crossover at each.

Neutral IS Values command (Calibration menu)

On the JEOL, this command will cycle through the magnifications and measure the neutral settings for image shift at each magnification.  Once these neutral settings are calibrated, they will be used as a base for image shift.  The program will display image shift relative to the neutral value.  When magnification is changed, the program will try to compensate for a change in neutral value and keep the actual image shift constant.

Camera Timing command (Calibration menu)

This command will take a series of pictures to determine the timing parameters for the dual shuttering mode of operation with a Gatan camera.  The procedure measures the mean counts in full-field images binned to 1024x1024 pixels, with a relatively long exposure (default 0.4 seconds).  Before running the procedure, it is necessary to adjust the beam so as to give moderately high counts in such an exposure without saturating the camera.  This may require a relatively dim beam.

When the operation is started, the program first takes several reference pictures with the DigitalMicrograph standard shuttering mode.  It then takes many (100 by default) pictures after adjusting the timing parameters so that the extra beam blanking will intrude on the exposure; the counts in such pictures indicate how long this blanking occurred during each exposure.  From the range of values, the program estimates values for three properties for this camera: StartupDelay, MinimumDriftSettling, and ExtraBeamTime.  The exposure time and the delay time before turning off the blanking are selected so that the blanking ends sometime during the exposure; if this fails to occur, the program will recommend rerunning the procedure with longer times.

Shutter Dead Time command (Calibration menu)

This command will take a series of pictures with very short exposure times to determine the dead time of the beam shutter, namely the amount of exposure lost.  The calibration is needed on JEOLs because the dead time can be ~0.01 sec, and this can lead to incorrect shutter time changes in routines that need to change the shutter time to reduce specimen exposure.  Before running it, set the Trial exposure to 0.1 second and adjust the beam and binning to give a moderate number of counts (1/4 – ½ saturation).  Start the routine and accept the default for the starting exposure time.  The program will take 7 pictures with progressively longer exposures and fit a line to determine the exposure that would give no counts.  It will then redo the procedure, starting at 0.01 sec above this exposure level.  At the end it will recommend an entry for ShutterDeadTime in the properties of the camera in question.

When there is a dead time property, the program will use this in estimating how to change an exposure time when going to a lower magnification, which it may have to do if the beam intensity cannot be lowered enough.  The value is also used when computing the dose resulting from camera exposures.

Administrator command (Calibration menu)

This command will toggle “Administrator mode”, which will enable you to save calibrations.  Administrator mode will also enable some procedures to make diagnostic outputs to message boxes or to the Log Window.

Save Calibrations command (Calibration menu)

This command will save the calibrations currently in the program to the system calibration file (SerialEMCalibrations.txt, in the system folder defined in your settings file).  The existing file will be renamed to SerialEMCalibrations.bak.  As a slight protection against accidental saving of this file, you must activate Administrator mode to enable this command.  In Administrator mode, the program will ask whether you want to save any changed calibrations before exiting.

Set Debug Output command (Calibration menu)

Use this command to change the key letters for debugging output, which are specified by the entry for DebugOutput in the properties file.  Any entry besides 0 will activate debugging output.  1 is used to get general output, while other letters give additional output: i for image shift-related items, l (lower case L) for low dose, L for more verbose low dose, u for update items when polling, e for event reports, and J for complete listing of JEOL calls.


Focus menu commands

The Focus menu offers the following commands for controlling Autofocus:

Measure Defocus	Measures current defocus.
Autofocus	Autofocus to the defined target value.
Move Focus Center	Shifts center of focus from display point to center of field.
Check Autofocus	Assess ability to autofocus by measuring defocus at 3 levels
Report shift & drift	Does focus detection and reports shift and drift.
Report On Existing	Reports shift and drift from pictures already present in buffers A-C.
Set Target	Enter target defocus to change to after autofocus.
Set Beam Tilt	Set amount of beam tilt for detecting defocus.
Set Threshold	Set threshold focus change for reiterating the autofocus.
Set Offset	Set focus offset to apply before measuring defocus
Drift Protection	Use three autofocus pictures to protect against drift
Reset Defocus	Set the defocus shown in the microscope panel to zero
Verbose	Outputs measured defocus to Log Window if it is open.

Measure Defocus command (Focus menu)

This command is used to run the autofocus routine for measuring defocus, without changing to the target defocus.  Upon completion, the program will inform you of the measured defocus and ask if you want to set the target defocus to this value.  This procedure is typically used if one  adjusts focus manually to give the desired image appearance, then measures defocus and uses the measured value to set the target.

Autofocus command (Focus menu)

This command will run the autofocus routine to measure defocus and then change focus to achieve the target defocus.  If the indicated change in focus is greater than a preset threshold, then the program will change focus by at most twice the threshold amount, and repeat the autofocus routine.  This threshold is set with the Set Threshold command described below.

Shortcut Key:	CTRL+G

Move Center command (Focus menu)

This command can be used at high tilt to change the focus so as to move the visible center of focus by a desired amount.  First click the left mouse button to set a display point at a place in the image with the desired focus.  Then select this command.  The center of the field in the next acquired image should be at the desired focus level.

Check Autofocus command (Focus menu)

This command can be used to assess how well autofocus will work.  The program will measure defocus using the current parameters at the current defocus, and at +5 and -5 microns away from that defocus.  It will then inform you of the measured defocus change as a fraction of the actual change in defocus in each direction.  If these numbers are greater than 0.9, autofocusing will be fairly accurate even for relatively large changes.  If the numbers are sufficiently large, autofocusing should work adequately during a tilt series; otherwise it will be unreliable.

Report Shift & Drift command (Focus menu)

Use this command to run the routine for measuring defocus and receive a report of the beam tilt-induced image shift plus the shift due to drift, all in pixels (unbinned pixels are reported if the images were binned).  Drift protection must be selected to get a meaningful estimate of drift.

Report on Existing command (Focus menu)

Use this command to measure the beam tilt-induced image shift and the shift due to drift from pictures that are already in buffers A-C from a previous autofocusing operation.  If drift protection is off, only the shift between pictures in buffers A and B will be reported.

Set Target command (Focus menu)

This command is used to enter a specific target defocus for autofocusing.  After the autofocus routine measures a defocus, it will change the focus setting so as to achieve this target level.  Selecting this command brings up a dialog box for entering the number; the default value shown is the current target.  Enter a defocus in microns.

Set Beam Tilt command (Focus menu)

This command is used to set the beam tilt that is used for autofocusing.  Selecting this command brings up a dialog box with a default value equal to the current beam tilt value.  The units are milliradians (mrad).

Set Threshold command (Focus menu)

Use this command to set the threshold for repeating the autofocus procedure after a large change in defocus.  The current value of the threshold is the default value shown in the dialog box.  Whenever autofocusing changes the focus level by more than this amount, the program will repeat the autofocus operation.

Set Offset command (Focus menu)

Use this command to set a focus offset that will be applied before any of the autofocusing operations invoked in the Focus menu.  For example, if the offset is set to +5 µm, then when you choose to measure defocus, the program will change defocus by +5 µm, take pictures for measuring defocus, restore the defocus by -5 µm, and subtract 5 µm from the measured value to determine the current defocus.  This option provides a way to autofocus to a target far from zero when the program is having difficulty autofocusing.

Drift Protection command (Focus menu)

Use this command to toggle between having two or three pictures taken for autofocusing.  With three pictures, the program can separate shift due to drift from the beam tilt-induced image shift used to determine defocus.  The measured defocus will thus be accurate if the drift is constant.

Reset Defocus command (Focus menu)

Use this command to reset the defocus readout in the Microscope Status panel to zero, without actually changing microscope focus.  On the Tecnai, this simply does the same thing as the Reset Defocus command in the Tecnai User Interface.  On the JEOL, the defocus is set to zero when the program starts and this is the only way to change it.

Verbose command (Focus menu)

This command toggles having the measured defocus value reported to the Log window, if it is open.


Macro menu commands

The Macro menu offers the following commands for controlling macros:

Stop	Stops macro at next command.
End	Stops macro at end of loop or repeat.
Resume	Resumes macro at place where it stopped.
Controls	Opens dialog box to set conditions for stopping a macro.
Edit 1, 2, ...	Opens a window for editing a macro.
Run 1, 2, ...	Runs a macro.

About Macros

A macro is a sequence of commands that SerialEM can execute.  A macro can perform all of the individual actions involved in a acquiring a tilt series, such as tilting, autofocusing, autoaligning, and saving images.  In addition, there are many commands for other actions such as moving the stage or changing magnification or defocus.

Macros can automatically repeat themselves and they can contain internal loops that are executed a specified number of times.  A macro can contain a statement at its end to switch to running another macro.  In addition, one macro can call another like a subroutine; when the second macro finishes, the next command in the calling macro will be run.  Neither loops nor calls may be nested more than one level deep.

The commands are typed into a Macro Editing Window.  One command is entered per line.  Blank lines and lines starting with # are ignored.  Comments can also be placed after the command on a line, starting with #.

Macros can be run either from the Macro menu or from the Camera & Macro Control Panel.  In addition, CTRL F1 through CTRL F10 are hotkeys for running macros.

Macros will be saved when Settings are saved, and when a settings file is loaded, any macros in that file will replace macros already in the program.

Macro commands

The macro processor is case-insensitive; you can enter commands as ALLCAPS

or alllowercase, but for readability it is helpful to use MixedCase as in the table.

Repeat	at end of macro to repeat it
Loop #	loop the given # of times until EndLoop
EndLoop	to end loop
DoMacro #	switches to given macro #
CallMacro #	calls other macro and returns
U or TiltUp	Tilt up by preset increment
D or TiltDown	Tilt down by preset increment
TiltTo #	Tilt to given angle
TiltBy #	Change tilt by the given amount
V or View	Acquire image with "view" parameters
F or Focus	Acquire image with "focus" parameters
T or Trial	Acquire image with "trial" parameters
R or Record	Acquire image with "record" parameters
L or Preview	Acquire image with “preview” parameters
M or Montage	Acquire montage
A or Autoalign	Align buffer A to autoalign buffer
AlignTo buffer_letter	Align buffer A to given buffer (e.g. B)
G or Autofocus [#]	Autofocus; if optional # is -1, just measure defocus without changing
S or Save	Save image
Copy b_l1 b_l2	Copy from b_l1 to b_l2 (e.g., Copy C A)
ChangeFocus #	Change focus by the given value in microns
SetMag  #	Set the mag to an actual film mag value
ChangeMag  #	Change mag by given number of mag steps
ReportMag	Report the current mag
SetSpotSize  #	Set the spot size to the given number
Delay #	Wait for given number of seconds (or milliseconds, if # > 60)
Pause [text]	Print option text and ask user whether to proceed
ImageShiftByPixels #X #Y	Change image shift by given # of pixels in X and Y
ImageShiftByUnits #X #Y	Change image shift by given # of IS units in X & Y
ReportAlignShift	Report the alignment shift of current image in A
ReportAccumShift	Report sum of shifts given by ReportAlignShift
ResetAccumShift	Reset the sum of shifts given by ReportAlignShift to 0
MoveStage #X #Y #Z	Move stage by micron increments in X, Y, Z
ReportStageXYZ	Report current stage position
ReportTiltAngle	Report current tilt angle
ReportMeanCounts [buf] [#]	Report the mean counts of the A buffer, or of the buffer indicated by ‘buf’; add a 1 to get unbinned counts per second
Echo  text	Print the text to the log window
ClearAlignment	Clear the alignment shift of the image in buffer A
ResetImageShift	Reset image shift and move stage to compensate
ResetShiftIfAbove #	Reset image shift and realign image if image shift is greater than the given # in microns
Eucentricity [#]	Refine eucentricity, or if optional # is entered, do rough eucentricity only (1), refine only (2), both steps (3), or refine and realign (6)
WalkUpTo #	Tilt up to given angle in steps and track image position
ReverseTilt [#]	Work out backlash and realign image after reversing tilt direction; enter optional 1 or -1 to specify a specific direction for further tilts
SetIntensityForMean #	Adjust beam intensity to yield the given number of counts, based on counts in the tilt-foreshortened area of the image in A
SetIntensityByLastTilt	Adjust beam intensity by the change in cosine of the tilt angle from the last tilt change
ChangeIntensityBy  #	Change beam intensity by the given factor using calibrations
SetPercentC2 #	Set the C2 lens strength to the given percentage
IncPercentC2 #	Change the C2 lens strength percentage by the given amount
ReportClock	Report seconds elapsed since last ResetClock command
ResetClock	Reset the time reported by ReportClock to 0
SetSlitWidth #	Set energy filter slit width in eV
SetSlitIn  [#]	Insert the slit; or if # is entered, insert if 1 or retract if 0
SetEnergyLoss #	Set the energy loss to the #
ChangeEnergyLoss #	Change the energy loss by the #
RefineZLP	Report time of day and run Refine Zero loss peak procedure
SetExposure #S #E [#D]	Set exposure and drift settling for exposure set S (0=view, 1=focus, 2=trial, 3=record, 4=preview) to the given values E and D.  The change will not survive opening the camera setup dialog.

Stop command (Macro menu)

Use this command to stop a running macro at the next step.

End command (Macro menu)

Use this command to stop a running macro at the end of a loop or the end of the macro (before repeating).

Resume command (Macro menu)

Use this command to resume a macro that was stopped with the Stop or End command.  The next command in sequence will be executed.  If you want to start a macro from the beginning, run it rather than resuming it.  In some cases a macro that stops because of an error condition can be resumed from the point at which it stopped.

Controls command (Macro menu)

Use this command to open the Macro Controls dialog boxin order to set conditions for stopping a macro.  The macro can be stopped when the stage reaches a certain tilt angle, image intensity falls below a certain level, image shift exceeds a certain amount, montage error is too large, or the macro has repeated a given number of times.

Edit command (Macro menu)

Use one of these commands to open a Macro Editing Window for editing the given macro.

Run command (Macro menu)

Use one of these commands to run a selected macro.


Tasks menu commands

The Tasks menu offers the following commands:

Set Intensity	Sets beam intensity based on current image mean.
Move Beam	Moves beam based on center marked by display point.
Eucentric - Rough	Finds eucentric point using coarse search only.
Eucentric - Fine	Refines eucentricity with pictures at a series of tilts.
Eucentric - Both	Finds eucentric point with coarse search then refines it.
Refine & Realign	Refines eucentricity and realign to original position.
Set Tilt Axis Offset	Set offset to center image shift on tilt axis
Reset IS & Realign	Resets Image Shift and realigns image position.
Set Iteration Limit	Set minimum Image Shift for reiterating Reset & Realign
Use Trial In LD	Use Trial area in Low Dose mode for Reset & Realign
Walk Up	Goes to high tilt in steps, tracking current position.
Walk Up & Anchor	Goes to high tilt in steps, leaving an image at intermediate tilt.
Reverse Tilt	Reverses tilt direction and realigns image position.
Verbose	Outputs progress and results of tasks to Log Window.

Set Intensity command (Tasks menu)

Use this command to change the beam intensity so as to achieve a desired mean level in images taken with Record parameters.  When you select this command, the program first computes the zero-tilt mean (mean of the tilt-foreshortened area) of the image in buffer A.  It then takes account of the exposure time and binning of that image to determine what the zero-tilt mean of a Record image would have been at the current beam intensity.  The program presents a dialog box in which you can enter the new intensity; the default value in this box is the computed mean for a Record image at the current intensity.  This value will give you some idea of how big a change you are requesting.  If you accept this default, nothing will change.  Enter the desired mean level.

Since the program changes intensity by condensing the beam, it is possible that the beam will become too small to cover the camera area.  If you increase the intensity by a substantial amount, you should check whether the beam still covers the area before going on to tasks that rely on full exposures.

Move Beam command (Tasks menu)

Use this command to move the beam by a controlled amount.  You can use it to center the beam by first taking a picture with the beam condensed enough to show much of its boundaries.  Then click the left mouse button to set a display marker point in the center of the beam.  Next, select this command, and the center of the beam will be moved to the center of the field.  In general, the beam will be moved by the amount that would move the marker point to the center of the field.

After you use this command, you need to take another picture to see the position of the beam.

Eucentric - Rough command (Tasks menu)

This command can be used to adjust the Z height to the eucentric point when the Z height might be off by more than 5-10 microns, which can often be the case when the specimen rod has just been inserted.  This procedure will find eucentricity to within about 0.5 micron.  This is good enough for tuning the microscope but not as accurate as the adjustment provided by the Eucentric – Fine command, which should be run before starting a tilt series.

This procedure does not make any attempt to keep the starting specimen location in the field of view.

The rough eucentricity procedure first switches to a low magnification (or, in low dose mode, it uses the parameters of the View area).  It takes pictures to determine how far the specimen moves laterally upon tilting, starting with a very small tilt increment and working up to a total increment of 8 degrees.  It may adjust the Z height more than once before finishing.

Eucentric - Fine command (Tasks menu)

Thus command can be used to adjust the Z height to the eucentric point when it is already within 5-10 microns.  It is thus a suitable procedure to use if you have moved to a new location on the specimen grid since the last time eucentricity was adjusted.  It is recommended that you run this command before starting a tilt series.

This procedure does not make any attempt to keep the starting specimen location in the field of view.  It can be run when not at zero tilt, and the program will return the stage to your pre-existing tilt at the end.

The fine eucentricity procedure first switches to a low magnification (or, in low dose mode, it uses the parameters of the View area).  It then takes pictures at a set of tilts between -24 and + 24 degrees.  From the image displacements during this series, it can compute both the distance in Z from the eucentric point and the lateral displacement of the tilt axis from the optical axis.  If image displacements become too large, the program will adjust Z height then repeat the procedure from the beginning.

Eucentric - Both command (Tasks menu)

Use this command to run the rough eucentricity procedure followed immediately by the fine eucentricity procedure.  This sequence does make any attempt to keep the starting specimen location in the field of view.

Refine & Realign command (Tasks menu)

Use this command to run the fine eucentricity procedure while retaining an item of interest centered in the field of view.  The program will first take a low-magnification reference picture, then do the fine eucentricity procedure, then take another picture at the original tilt angle and use that to restore the alignment.  In low dose mode, the reference picture will be taken of the View area.  It is not necessary to be at zero tilt to run this command.

Set Tilt Axis Offset command (Tasks menu)

Use this command to set the offset of the tilt axis from the optical axis, which is used to keep image shift centered on the optical axis.  If the tilt axis is more than a micron from the optic axis, it is desirable to set this offset to minimize specimen movements in Y and Z during tilting.  The offset should be set as a system-wide property.  However, if this property setting is not adequate, or if the offset varies significantly from place to place, this command can be used to set the offset.  It is placed in this menu because the lateral offset from the Refine Eucentricity procedure indicates how much the offset should be changed.  When the command is selected, a dialog box comes up informing you of the current tilt axis offset, and the total offset implied by the last run of Refine Eucentricity, if any.  The default value in the box is the implied total offset (or just the current offset, if Refine Eucentricity has not been run.)  A tilt axis offset has an effect only if you have selected ‘Center image shift on tilt axis’ in the Image Alignment and Focus control panel.

Reset IS & Realign command (Tasks menu)

Use this command to reset the microscope image shift (IS) while retaining an item of interest centered in the field of view.  The program will take a reference picture, reset the image shift to zero and move the stage to compensate, then take another picture and align it to the reference picture.  At the end, image shift will no longer be zero because of this alignment; in fact, it can be as much as one-quarter of the original image shift because of inaccuracy in stage movement.  The reference picture will be taken at low magnification or, if in low dose mode, with the View parameters.

If you just want to reset image shift without having a feature stay centered, use the Reset Shifts button in the Image Alignment & Focus panel .

Set Iteration Limit command (Tasks menu)

Use this command to change the criterion that the Reset IS & Realign procedure uses to decide whether to reset the shift and realign a second time.  After resetting the shift, the program takes an image and aligns it to image taken before the reset.  If image shift is still greater than this criterion, it will be reset again, and another image taken and aligned.

Use Trial in LD command (Tasks menu)

Toggle this entry to switch between using the View and Trial areas for the Reset IS & Realign procedure.  The View area is the default because the stage movement involved in resetting the shift is unpredictable, and the larger field of view of the View area is assumed to be needed to do the reset reliably.  It may be safe to use the trial area if your limit for resetting image shift is small (~1 micron), if the field of view of the trial area is fairly large (2 microns), and if your stage is adjusted so that stage movements are reliable at high tilt for the holder being used.

Walk Up command (Tasks menu)

Use this command to go from one tilt angle to another while retaining an item of interest centered in the field of view.  Typically this would be used to go from zero tilt to a high tilt angle.  After you select the command, you will encounter a dialog box in which to enter the destination tilt angle.

This procedure involves taking alignment pictures at a series of tilt angles, with a tilt interval that is quite coarse at low angles and finer at high angles.  Image shift will be reset whenever it gets too high, but no autofocusing is done.  The pictures are taken with the Trial parameter set and, if in low dose mode, the pictures are of the Trial area.  If the magnification is too high for reliable tracking, the pictures will be taken at a lower magnification.

If you choose a destination angle that is in the opposite direction from the last direction of tilt, the routine will run the Reverse Tilt procedure first to avoid losing the area of interest.

Walk Up & Anchor command (Tasks menu)

Use this command to do the same operation as in the Walk Up command but also leave an image behind at a specified intermediate tilt angle.  The image will be left in the last image buffer and can be used as an “anchor” for alignment during a tilt series.

Reverse Tilt command (Tasks menu)

Use this command to reverse the direction of tilting while retaining an item of interest centered in the field of view.  For example, if you tilt up to a high angle, center your item of interest, then try to start a tilt series by just tilting back down, the specimen may move considerably because of backlash in the stage.  This problem can be avoided by using this command.  The program will take a reference picture, tilt far enough in the current direction to work out the backlash, tilt back to the starting angle, then take another picture and align it to the reference picture.  The reference picture will be taken at low magnification or, if in low dose mode, with the View parameters.

Verbose command (Tasks menu)

Use this command to toggle diagnostic output to the Log window from various the various procedures run from the Tasks menu.


Tilt Series menu commands

The Tilt Series menu offers the following commands:

Setup/Start	Open dialog box to set parameters for a tilt series.
Terminate	Ends tilt series and returns full program control to user.
Stop	Stops all actions as soon as possible.
End Loop	Stops at end of loop.
Back Up	Opens dialog box to back up to a previous tilt angle.
Resume	Opens dialog box to resume series after potential changes.
One Loop	Does one loop of the tilt series.
One Step	Does one step of the tilt series.
Verbose	Outputs predictions and actions to Log window
Autosave Log	Toggles saving the Log Window to a file after every tilt.
Debug Mode	Toggles sending debugging output to Log Window.

Setup/Start command (Tilt Series menu)

Use this command to open the Tilt Series Setup dialog box to set the parameters for a new tilt series and start the series, or to adjust the parameters of one that is running.  If a tilt series is already running, you will have the option of resuming the series at the next step in its sequence, or of leaving the dialog box and resuming the tilt series at another point through the Tilt Series Resume dialog box .

Terminate command (Tilt Series menu)

Use this command to terminate a tilt series and restore all program controls to their normal state.  The Tilt Series Controller will no longer control the various buffer and saving options in the program.  You will have the option of leaving your image file open or closing it.

Stop command (Tilt Series menu)

Use this command to stop a tilt series at the next logical step.  The Tilt Series Controller will still be in control of the program.  If you open the the Tilt Series Resume dialog box you will be able to see exactly where the series stopped, because the dialog box contains a statement of the next action to be performed.

End Loop command (Tilt Series menu)

Use this command to stop a tilt series after all actions have been completed at the current tilt angle.  The Tilt Series Controller will still be in control of the program.

Back Up command (Tilt Series menu)

Use this command to open the Tilt Series Back Up dialog box in order to back up to a previous tilt angle and redo operations from that point.  You can use this procedure more than once, if necessary, before selecting the point from which you want to resume.  When you back up, the Tilt Series Controller will try to set up an appropriate reference image for the new tilt angle.

Resume command (Tilt Series menu)

Use this command to open the Tilt Series Resume dialog box .  This dialog box will give you a chance to change the next action to be performed.  You can choose to redo some actions at the current tilt angle, or, to a limited extent, you can skip some actions that you have performed manually.

One Loop command (Tilt Series menu)

Use this command to perform one loop of a tilt series.  The Tilt Series Controller will resume from the point at which it was stopped and will go on until all operations are complete at a tilt angle.

One Step command (Tilt Series menu)

Use this command to advance one step of the tilt series.  There are many more steps in the loop of the Tilt Series Controller than actual actions under particular conditions, so in many cases nothing will appear to happen when this command is selected.

Verbose command (Tilt Series menu)

Use this command to toggle output of tracking and prediction information and other status messages to the Log window during a tilt series.  If this item is not checked, then only the summary information about each image saved will be printed.

Autosave Log command (Tilt Series menu)

Use this command to toggle automatic saving of the Log Window to a file after every tilt.  If the Log Window has not yet been saved to a file, you will be prompted to specify an output file after the first tilt, but thereafter this file will be continuously updated.

Debug Mode command (Tilt Series menu)

Use this command to toggle output to the Log Window of numerous state variables in the Tilt Series Controller.  The output will occur before each action.


Process menu commands

The Process menu offers the following commands:

MinMax/Mean	Computes min, max, mean and S.D. of active image.
Zero-tilt mean	Computes mean of area foreshortened by tilting.
Set Intensity	Sets beam intensity based on current image mean.
Move Beam	Moves beam based on center marked by display point.
FFT	Computes full-sized Fourier Transform of image in Buffer A
Binned FFT	Computes FFT of image binned to 512 or binned by 2.
Rotate Left	Rotates image in Buffer A counterclockwise.
Rotate Right	Rotates image in Buffer A clockwise.
Fix Dark X-rays	Removes X-rays from image in Buffer A using dark criteria
Set Dark Criteria	Allows you to set criteria for dark X-ray removal
Fix Image X-rays	Removes X-rays from image in Buffer A using image criteria
Set Image Criteria	Allows you to set criteria for image X-ray removal
Show Cross-corr	Show cross-correlation from the current autoalignment

Min/Max/Mean command (Process menu)

Use this command to obtain the minimum, maximum, and mean values of the image in the active window.  The information will appear in a message box, or in the Log window if it is open.  The computation is done on the active window, which may not be the main window.

Shortcut Key:	CTRL+I

Zero-tilt mean command (Process menu)

Use this command to obtain the mean value of the approximate area in the current buffer that corresponds to the full field at zero tilt.  For example, if the tilt is 60 degrees, the magnification is such that the tilt axis is near vertical, and the image is 1024 pixels wide, then the mean will be taken of the central half of the image with X between 512 and 768.  Note that the mean is taken of the current buffer in the main window, even if it is not the active window.

FFT command (Process menu)

Use this command to take a full-sized Fourier transform (FFT) of the image in buffer A.  The FFT will be placed into buffer A and existing images will be rolled to higher buffers to the extent specified in the Buffer Control Panel, just as when a new image is acquired.

Binned FFT command (Process menu)

Use this command to take a binned FFT of the image in buffer A.  Before taking the FFT, the image will be binned down to 512 by 512, or by a factor of two if it is already this small.  The FFT will be placed into buffer A and existing images will be rolled to higher buffers to the extent specified in the Buffer Control Panel, just as when a new image is acquired.

Rotate Left command (Process menu)

Rotate Right command (Process menu)

Use these commands to rotate the image in buffer A counterclockwise (left) or clockwise (right) by 90 degrees.  The rotated image will be placed into buffer A and existing images will be rolled to higher buffers to the extent specified in the Buffer Control Panel, just as when a new image is acquired.

Fix Dark X-rays command (Process menu)

Use this command to apply the X-ray removal routine to the image in Buffer A, using the criteria for removal of X-rays from dark references.  The image with X-rays removed will be placed in Buffer A, and existing images will be rolled to higher buffers to the extent specified in the Buffer Control Panel, just as when a new image is acquired.

Set Dark Criteria command (Process menu)

Use this command to set the criteria for dark reference X-ray removal.  First you will encounter a dialog box asking for a criterion for the absolute amount above the mean, then another dialog asking for a criterion for the number of standard deviations above the mean, then a final dialog asking whether a pixel must satisfy both criteria or just one of them.  These criteria replace the ones specified in the property file and will apply only during this session of the program.

Fix Image X-rays command (Process menu)

Use this command to apply the X-ray removal routine to the image in Buffer A, using the criteria for removal of X-rays from images.  The image with X-rays removed will be placed in Buffer A, and existing images will be rolled to higher buffers to the extent specified in the Buffer Control Panel, just as when a new image is acquired.

Set Image Criteria command (Process menu)

Use this command to set the criteria for X-ray removal from images, which is activated for a particular camera with the ShowRemoveXRaysBox property and for individual camera parameter sets with the Remove X rays checkbox in the Camera Setup dialog box.  First you will encounter a dialog box asking for a criterion for the absolute amount above the mean, then another dialog asking for a criterion for the number of standard deviations above the mean, then a final dialog asking whether a pixel must satisfy both criteria or just one of them.  These criteria are camera-specific; they are stored in your settings file and will persist between sessions of the program.

Show Cross-corr command (Process menu)

Use this command to show the cross-correlation used to autoalign the image in buffer A to the image in the autoalign buffer.


Navigator menu commands

The Navigator menu has commands for opening the Navigator window, operating on Navigator files, and performing less common Navigator operations.

Open	Open the Navigator window.
Read & Open	Read a Navigator file and open Navigator window if it is not open.
Save	Save current Navigator items to a file.
Save As	Save current Navigator items to a different file.
Close	Close the Navigator window.
Autosave	Periodically save entries to file if one is open.
Setup Corner Montage	Define a montage that captures area inside corner points.
Setup Polygon Montage	Define a montage that captures area inside a polygon.
Setup Full Montage	Define a montage to capture the full grid area.
Aligned Super Montage	Add polygons representing overlapping montages aligned to camera axes
Skewed Super Montage	Add polygons representing overlapping montages aligned to specimen axes
Transform Points	Transform items to the current registration.
Delete Item	Delete the current item.
New Map from Image	Make a new map from the current image.
Acquire Areas	Acquire sequence of areas automatically.
Convert Maps to Bytes	Convert maps to bytes when creating or loading them to save memory.

Open command (Navigator menu)

Use this command to open the Navigator window.

Read & Open command (Navigator menu)

Use this command to read in a Navigator file as well as open the window if it is not already open.  The file will become the current file for saving into.

Save command (Navigator menu)

Use this command to save Navigator items to a file.  The file is a simple tab-delimited text file with the properties of each item.  If there is no current Navigator file, you will enter the Save As dialog box to specify the file, which will have the default extension .nav.  When you select this command again, the entries will be saved to this file.

Save As command (Navigator menu)

Use this command to save Navigator items to a file different from the current file.  You will enter the Save As dialog box to specify the file.  The new file will become the current file for saving into.

Close command (Navigator menu)

Use this command to close the Navigator window.  If the contents of the window have changed since it was last saved, you will be asked if you want to save them to a file.

Autosave command (Navigator menu)

Toggle this menu item on to have the Navigator items saved to a file periodically if they have changed.  If there is no currently open file, this option has no effect.

Setup Corner Montage command (Navigator menu)

Use this command to set up a montage to capture the area within the current set of corner points.  Be sure to go to the desired magnification before activating this command.  You will be opening a new image file for the montage, so if there is already a file open the program will ask whether you want to close it.  The program will compute the number of montage frames in X and Y required to capture the area, and decide whether the montage should be acquired with image shift or stage movements.  If the area of the Record parameter set is substantially smaller than the camera field, you will be asked whether you want the frame size to be kept smaller than the current Record area.  You might want this if the beam is too small to fill the whole camera field.  Otherwise, the program will make the frames as large as possible.  Unless there are too many frames, you will then go on to the Montage Setup dialog box, in which you will see the number of frames and their size and overlap.  If you change any of these parameters, the acquired area may no longer match what was specified.  If the montage is reasonable, go on to the File Properties dialog box then the Save As dialog box.

After the montage is defined, the program will compose a list of pieces that are not needed to capture the intended area.  These pieces will be excluded when the montage is acquired.

Setup Polygon Montage command (Navigator menu)

If the current Navigator item is a polygon, use this command to set up a montage to capture the area within the polygon.  It works the same as the Setup Corner Montage command .

Setup Full Montage command (Navigator menu)

Use this command to set up a montage to capture the whole grid area.  A set of points will be composed that contain the grid area.  The command then works the same the Setup Corner Montage command .

Aligned Super Montage command (Navigator menu)

Use this command to add a set of rectangles at the positions of overlapping montages, laid out on a rectangular grid that is aligned to the camera axes.  First, you must have montaging set up with the desired number of pieces and magnification.  Specify a center position in the active image by clicking with the left mouse button.  Then select this command.  You will be asked to enter the number of montages in X and in Y then the number of pixels of overlap in X and Y.  A set of polygons will be created, numbered by their positions in the super montage.  To try again with different parameters, just push the Delete Item button repeatedly.

Skewed Super Montage command (Navigator menu)

Use this command to add a set of rectangles at the positions of overlapping montages, laid out on a grid that is aligned to the specimen axes.  This array will appear staggered on a camera image, but after the montages are individually rotated so that the tilt axis is vertical, they will lie on a rectangular grid.  Other than this, the command operates the same as the Aligned Super Montage command .

Transform Points command (Navigator menu)

Use this command to transform items from one or more other registrations to the current registration.  The program will find a transformation whose complexity depends on the number of registration points available.  Just a shift will be found with 1 point, a rotation and shift with 2 points, rotation, scaling, and shift with 3 points, or a full linear transformation including stretching with 4 points.  All items except the registration points will then be transformed and associated with the current registration.  The program will give a message about how well the points fit the transformation equation.  If the fit is very poor, you can undo the transformation by changing the current registration number back to the previous value and redoing this command.

Delete Item command (Navigator menu)

Use this command to delete the current item from the list.

New Map from Image command (Navigator menu)

Use this command to define the current image as a map.  A map consists of an image that has been saved to a known file.  There must be an open file to create a map.  If it has single frames, the image in buffer A must have been saved to the file.  If the file is a montage, the image in Buffer B must be an overview from a montage capture.  It is possible to use an image or montage overview read in from a file to make a map, but this is discouraged because of the potential ambiguity about the stage position at which they were taken.

Acquire Areas command (Navigator menu)

Use this command to acquire a series of areas at the points marked for acquisition with the Acquire check box.  There must be an image file open before the command can be used.  If montaging is to be used, this means that the montaging must be set up already.  Be sure to have magnification, intensity, and exposure times set before starting the acquisition.

As each area is acquired, it will be added as a map item with a label based on the label of the point being acquired (e.g., 12-A for point 12).  The original point will no longer be marked for acquisition and it will no longer be drawn, to reduce clutter.  Once the acquisition is finished, you could delete the original points.

Convert Maps to Bytes command (Navigator menu)

This command toggles an option to keep maps in memory as bytes.  The option does not affect how maps are stored in the image file (typically as integers).  Byte conversion will reduce memory usage by 2/3, so that the memory required is just one byte per pixel.  The intensity scaling determined by the Black and White levels in the Image Display control panel will be associated with an image when a new map is created.  This scaling is then applied to convert the image to bytes, both when the map is defined and when the map is reloaded from file.  This scaling could make it impossible to see some light or dark image details.  There are two ways to prevent such scaling problems.  First, adjust the Black and White levels to avoid truncating image intensities before designating an image as a map.  Second, use this menu entry to turn off the conversion option, load the map, adjust the Black and White levels as desired, and turn the option back on.


Window menu commands

The Window menu offers the following commands, which enable you to manipulate multiple image windows in SerialEM:

New Window	Copies active image to a new image window
Cascade	Arranges windows in an overlapped fashion.
Tile	Arranges windows in non-overlapped tiles.
Resize Main	Resizes the main window to fill available space.
Window 1, 2, ...	Goes to specified window.
New Window command (Window menu)

Use this command to copy the active image into a new window.

Cascade command (Window menu)

Use this command to arrange multiple image windows in an overlapped fashion.

Tile command (Window menu)

Use this command to arrange multiple image windows in a non-overlapped fashion.

Resize Main command (Window menu)

Use this command to restore the main window to fill all of the space inside the SerialEM frame.

Shortcut Key	SHIFT+R.

1, 2, ... command (Window menu)

SerialEM displays a list of image windows at the bottom of the Window menu.  A check mark appears in front of the name of the active window.  Choose a window from this list to make it active.


Help menu commands

The Help menu offers the following commands, which provide you assistance with this application:

Help Topics	Opens help and provides table of contents.
Help Index	Offers you an index to topics on which you can get help.
Context Help	Allows you to get help on specific item by clicking on it.
Using Help	Describes ways of accessing help in SerialEM
About	Displays the version number of this application.
Help Topics command (Help menu)

Use this command to display the opening screen of Help.  From the opening screen, you can jump to instructions for using SerialEM and various types of reference information.

Once you open Help, you can click the Contents button whenever you want to return to the opening screen.

Help Index command (Help menu)

Use this command to jump to the top of the help file, where there are links to all of the main help topics.

Context Help command

Use the Context Help command to obtain help on some portion of SerialEM.  When you choose this command or push Shift F1, the mouse pointer will change to an arrow and question mark.  Then click somewhere in the SerialEM window, such as a control panel.  The Help topic will be shown for the item you clicked.

Shortcut

Keys:	SHIFT+F1

Using Help command (Help menu)

SerialEM incorporates many of the standard Windows Help features, but some things do not work in the ideal fashion.

The Help Topics command will take you to the opening window of the help system, in which you can select contents, index, or search functions.  Index functions will probably not work.

The Help Index command will take you to the top of the help file where there are links to all available topics.

In addition, there are several ways to activate context-sensitive help in SerialEM:

Press F1 when the mouse is over a menu entry.  You will go to the specific help for that item.

Press F1 when the mouse is over a dialog box.  You will go to the help for the entire dialog box.  F1 will not work when pressed over a control panel.

Press the ? button in a control Panel or dialog box.  You will go to the help for the whole panel or dialog box.

Press Shift F1 then click on in item in the program.  You will go to help for the item or for the window that the item is in.

About command (Help menu)

Use this command to display the copyright notice and version number of your copy of SerialEM.


File Open dialog box

The following options allow you to specify which file to open:

File Name

Type or select the filename you want to open.  This box lists files with the extension you select in the List Files of Type box.

List Files of Type

Select the type of file you want to open:

<< List your application's file types here. >>

Drives

Select the drive in which SerialEM stores the file that you want to open.

Directories

Select the directory in which SerialEM stores the file that you want to open.

Network...

Choose this button to connect to a network location, assigning it a new drive letter.


File Save As dialog box

The following options allow you to specify the name and location of the file you're about to save:

File Name

Type a new filename to save a document with a different name.  SerialEM adds the extension you specify in the Save File As Type box.

Drives

Select the drive in which you want to store the document.

Directories

Select the directory in which you want to store the document.

Network...

Choose this button to connect to a network location, assigning it a new drive letter.


Status Bar

The status bar is displayed at the bottom of the SerialEM window.  It cannot be hidden because it provides crucial information when the program is busy capturing an image or performing other time-consuming tasks.

The left area of the status bar describes actions of menu items as you use the arrow keys or mouse to navigate through menus.

The right areas of the status bar indicate what program action is underway.  The rightmost pane indicates image acquisition and blinks during this activity.  The middle pane typically indicates a task that may involve several image acquisitions.  The left pane provides message about the most complex tasks, running a tilt series or a macro.


Title Bar

The title bar is located along the top of a window.  It contains the name of the application and open image file.

To move the window, drag the title bar.  Note: You can also move dialog boxes by dragging their title bars.

A title bar may contain the following elements:

Application Control-menu button

Image window Control-menu button

Maximize button

Minimize button

Name of the application

Name of an open image file

Restore button


Scroll bars

Displayed at the right and bottom edges of a Log window or Macro Editing window.  The scroll boxes inside the scroll bars indicate your vertical and horizontal location.  You can use the mouse to scroll to other parts of the text in the window.


Size command (System menu)

Use this command to display a four-headed arrow so you can size the active window with the arrow keys.

After the pointer changes to the four-headed arrow:

1.	Press one of the DIRECTION keys (left, right, up, or down arrow key) to move the pointer to the border you want to move.

2.	Press a DIRECTION key to move the border.

3.	Press ENTER when the window is the size you want.

Note:  This command is unavailable if you maximize the window.

Shortcut

Mouse:	Drag the size bars at the corners or edges of the window.


Move command (Control menu)

Use this command to display a four-headed arrow so you can move the active window or dialog box with the arrow keys.

Note:  This command is unavailable if you maximize the window.


System Minimize Command
Minimize command (application Control menu)

Use this command to reduce the SerialEM window to an icon.

Shortcut

Mouse:	Click the minimize icon [image] on the title bar.

Keys:	ALT+F9


Maximize command (System menu)

Use this command to enlarge the active window to fill the available space.

Shortcut

Mouse:	Click the maximize icon [image] on the title bar; or double-click the title bar.


Next Window command (image window Control menu)

Use this command to switch to the next image window.  SerialEM determines which window is next according to the order in which you opened the windows.


Previous Window command (image window Control menu)

Use this command to switch to the previous image window.  SerialEM determines which window is previous according to the order in which you opened the windows.

Shortcut

Keys:	SHIFT+CTRL+F6


Close command (Control menus)

Use this command to close the active window or dialog box.

Double-clicking a Control-menu box is the same as choosing the Close command.

Shortcuts

Keys: 	ALT+F4 closes a dialog box or exits SerialEM


Restore command (Control menu)

Use this command to return the active window to its size and position before you chose the Maximize or Minimize command.


About Control Panels

SerialEM has many controls and status indicators arranged in a set of control panels to the left of the main image window.  Here are the common features of these panels:

A panel can be open or closed with the +/- button in the upper left corner.  When it is closed, you will still see the title and +/- button.

Many panels are divided into two parts, with the lower part containing less frequently used controls or option settings related to the main purpose of the panel.  This lower section can be opened and closed with the +/- button labeled ‘Options’.

A panel can be detached from the column of panels and turned into a free-standing window that can be positioned anywhere on the screen by pressing the ‘F’ button.  Once a panel has been floated, it can be docked back into the column by pressing the button again (now labeled ‘D’).

The ‘?’ button in the upper right corner will open the help page for the control panel.

The state of control panels will be saved with your Settings and restored when you restart the program (except the montage panel, which always starts closed).

When you click the mouse on a control or on the background of a control panel, the panel will almost always restore the keyboard focus to an image display window, so that the program will still respond properly to hotkey input.  There are a few pixels where this will not happen: the middle line of spin button controls, the periphery of the brightness/contrast sliders, and the title bar of a floating panel.  If SerialEM loses focus and its title bar goes gray, just click in the title bar or in the main image window.


Buffer Status Panel

This panel shows detailed information about the image buffer currently shown in the main display window, and summary information about several other buffers.  For an ordinary image acquired by the user, the summary line for a buffer will show what parameter set was used to acquire the image and whether it is saved to file or not.  Other descriptive terms will appear when the program has acquired an image for calibration or tracking, when an image has been processed, or when the image was composed by the montage acquisition routine.

Current buffer spin button

This control can be used to navigate through the image buffers.  The up arrow will advance to higher buffers (equivalent to pressing the PageDn key) and the down arrow will move back toward the A buffer (the same as pressing PageUp).

Current buffer details

Below the summary line for the current buffer will appear the image size in pixels, the binning used to acquire the image, the tilt angle, the X and Y stage positions, and the microscope defocus setting.  If the image is one piece of a montage, the piece number will be substituted for the X and Y stage positions.

Additional buffer summaries

Up to three additional buffer contents are described, starting from the first buffer and skipping the current buffer.


Buffer Control Panel

This panel contains controls for copying images from one buffer to another, saving images to file, and setting options that govern how buffers are treated during image acquisition and storage.

Copy Active Image to Buffer

This row of buttons will copy the active image to another buffer.  The active image is either the current image in the main window, or the image in a secondary window, if there is a secondary window that has the input focus (title bar highlighted rather than gray).  The first four buttons from the left will copy to buffers A through D, respectively.  The fifth button has a spin button control to allow you to dial up any other buffer in the program.  The ‘New’ button will copy the image into a new secondary image window.

SAVE A

This button will save the image in buffer A to a file.  If there is no image file open yet, you will first enter the File Properties and Save As dialog boxes to specify a file.  This file will become the open image file, available for further saving.

Save Active Image

This button will save the active image to a file.  The active image is either the current image in the main window, or the image in a secondary window, if there is a secondary window that has the input focus (title bar highlighted rather than gray).  If there is no image file open yet, you will first enter the File Properties and Save As dialog boxes to specify a file.  This file will become the open image file, available for further saving.

Roll Buffers spin button

This control governs how many images are preserved by being moved to higher buffers when a new image is acquired.  For example, if the panel says ‘Roll Buffers A -> C’, then when a new image is acquired, the image in A will be moved to B, the image in B moved to C, and the image in C deleted.  (An exception to this occurs when the image in A was acquired in a continuous acquisition.)  The up and down arrows will increase and decrease the number of images rolled through the buffers, respectively.

Copy on Save

Selecting this option will cause an image to be copied automatically to the buffer after the last rolling buffer when the image is saved to file.

Autoalign to B

Ordinarily, an image in buffer A is autoaligned to the image in the buffer after the last rolling buffer, which means that a reference image placed there will be preserved as new images are taken.  With this option selected, images will be aligned to buffer B instead, which allows successive images to be aligned to each other without copying one to another buffer.

Read Buffer spin button

This control governs what buffer images are placed into when they are read from a file.  If you never use this control, the Read Buffer will be maintained to be two buffers past the last rolling buffer, or three buffers when in low dose mode.  Thus, changing the number of buffers to roll, or going into or out of low dose mode, will generally change the Read Buffer.  However, if you select a different buffer to read into with this control, then these automatic changes may not occur.

Protect unsaved Record images

Selecting this option will protect images acquired with the Record parameter set from being deleted if they have not been saved to file.  You will have to confirm that you want to delete such an image.

Align on Save

Selecting this option will cause an autoalignment to be done whenever an image is saved to file.  This operation is crucial for minimizing the accumulation of alignment errors due to drift during a tilt series.  It makes the reference for aligning the next image be located where it was supposed to be (i.e., where it would have been if there were no drift), so that the desired alignment is preserved over the long term.

When this feature is active, you need to take special action to change the desired image position, otherwise any change that you introduce might be wiped out when you save.  The Tilt Series Controller takes care of this problem for you, but if you are doing a tilt series manually, you must impose a new alignment on the image in the Autoalign buffer.  There are two ways to do this.  One is to get an image in A that is in your new desired position, shifting it with the right mouse button if necessary, and then copy it to the Autoalign buffer.  The other is to display the Autoalign buffer and shift it to the desired position with the right mouse button.


Image Display Control Panel

This panel allows you to see and control how image intensities are displayed.  When an image is acquired or read from a file, pixels are sampled over a particular area to determine how to scale the intensities for display.  The program finds the lower and upper intensities beyond which only a certain small fraction of pixels have intensities, and stretches the contrast to map these limiting intensities to black and white on the screen.  These values are shown in the Black and White text boxes.  You can control the size of the area that will be analyzed, as well as the percent of pixels whose intensities will be saturated at each extreme.  You can also simply change the black and white values directly.  Nothing you do in this window will affect the pixel values that get saved to a file.

Zoom

The text box displays the image zoom and allows a zoom value to be entered directly.  The up and down arrows allow you to increase or decrease the zoom.

Black and White levels

These text boxes show the image intensities that correspond to black and white on the screen.  Because some intensities values are typically truncated, these are not the same as the minimum and maximum intensities in the image, which you can determine with the Min/Max/Mean command in the Process menu.  You can enter new values in these boxes to control the degree of saturation at the extreme values in the image.

Brightness and Contrast sliders

Use these sliders to adjust the brightness and contrast settings of the display for the current image.  Different images can have different brightness/contrast settings.  A newly acquired image will inherit its settings from the previous image in the A buffer.  If an image appears saturated at a uniform gray level after adjusting these sliders, you probably need to adjust the Black or White levels to see detail in these regions.  If a bright area appears saturated, try raising the White level; if a dark area is saturated, lower the Black level.

Set Truncation

Use this button to enter new values for the percent of pixels that will have their intensities truncated at the low and the high end of the intensity range.  You will enter two dialog boxes for entering new values.  The current image will be re-analyzed and displayed with the new Black and White levels, and the same percentages will apply for new images that are acquired.

Set Area Fraction

Use this button to enter a new value for the fraction of image to analyze when determining the Black and White levels.  Enter a fraction of the linear extent of the image.  For example, if you enter 0.5, then only pixels from 256 to 768 in X and Y in a 1024 x 1024 pixel will be analyzed, which is actually only one-quarter of the image area.

Autozoom

When this option is selected, the display zoom will change automatically when switching between images with different binning.  This allows full-size and binned images to be compared easily.

Zoom step selection

These radio buttons control whether zoom will change by fixed steps of 1.41, or to fixed values such as 1, 1.41, 2, etc.  Zoom is initially set automatically so that images fit in the main window, and is adjusted to maintain this fit whenever the program window is resized.  Having zoom change by fixed steps will keep the images fitting in the window when you zoom up and back down.


Microscope Status Panel

This window displays critical microscope parameters so that the Tecnai User Interface (TUI) can be covered over by SerialEM.

Screen current (na) and Float button

This readout shows the screen current in nanoamps (na).  If the small focusing screen is in, then this is the amount of current that would be measured if the beam uniformly covered the whole large projection screen.  If SerialEM thinks the small focusing screen is in it will display the label as na-fs.  This readout has some smoothing to prevent jitter, so it may not respond to very small changes in screen current.

The Float button will launch a small window with the screen current readout.  This window will stay on top of all other windows, so it can be seen even if TUI is brought on top of SerialEM.  Smoothing is optional in this readout, so it may be more suitable for use when aligning gun tilt.

Dose button

The Dose button will launch a small window showing a cumulative dose in electrons per square Angstrom.  Dose is added whenever the screen is down, and whenever a picture is taken, except that pictures of the Trial and Focus areas will be ignored in Low Dose mode.  No dose is added when the beam intensity is outside a calibrated range.  Press the Reset button to zero the cumulative dose.

Magnification (X)

This readout shows the current magnification.  It changes when the screen is raised, so it should match the readout in the TUI.

Defocus (Def)

This readout shows the same defocus value as appears in TUI.  It will NOT be the same as the “measured defocus” reported by the autofocus routine.  The defocus value shown here is based on the current objective lens strength, and it is relative to the lens setting when you last selected “Reset Defocus” in TUI.  The “measured defocus” is based on the amount of image displacement that occurs when the beam is tilted, and it should reflect how far the specimen is from zero defocus.

Image Shift (IS)

This readout shows the total image shift in microns.  When a montage is being acquired, this value will briefly reflect the image shift of individual pieces.  However, in low dose mode, it will show only the image shift used to select the field of view and will not show the additional image shift of the acquisition areas.

Objective Lens Strength (Obj)

This readout shows the objective lens strength in percent, and will match the value shown in TUI.

Vacuum Quality (VAC)

This is an indicator of vacuum quality that may alert you to watch the actual vacuum readings in TUI.  It should be set up so that green means near perfect, yellow means adequate for working, and red means do not open the column values.

Spot

This readout shows the spot size.


Tilt Control Panel

This window displays the current tilt angle, allows one to tilt by an increment or to go to a desired angle, and contains options for tilt-related parameters.

Tilt Angle Readout

This readout shows the current tilt angle, the same as the ‘A’ readout in the Tecnai User Interface.

Tilt Up

Tilt Down

These buttons will tilt toward positive or negative tilt angles by the current tilt increment, which is either the basic tilt increment, or this basic increment multiplied by the cosine of the tilt angle if ‘Cosine increments’ is selected.

Set Increment

Use this button to set the basic tilt increment.  A dialog box will appear, with the current value as the default.  Enter a value in degrees.

Set Delay

Use this button to set the delay to be applied after tilting by the basic increment.  A dialog box will appear, with the current value as the default.  Enter a value in seconds.  This delay time must expire before a new image is taken with the camera.

Cosine Increments

Use this button to select cosine tilt increments, in which the increment is proportional to the cosine of the tilt angle.


Camera & Macro Control Panel

This panel has buttons for taking images and controlling macros or actions during a tilt series.  Several of the buttons will change when the Tilt Series Controller is activated.

Setup

This button will open the Camera Setup dialog box and allow you to set parameters like exposure time, field of view and binning for the 5 different exposure modes

View

Focus

Trial

Record

These buttons will acquire an image using the View, Focus, Trial, or Record parameters.

Preview/Montage

Ordinarily, this button will acquire an image with the Preview parameter set.  If montaging has been started, the button will acquire a montaged image.

End

If a macro is running, this button will end a macro at the end of a loop or at the end of the macro.  If the Tilt Series Controller is active, the button will stop the series after completing all actions at the current tilt angle.

Resume

If a macro has been running, this button will resume the macro at the next step.  If the Tilt Series Controller is active, the button will open the Tilt Series Resume dialog box

STOP

This is general Stop button for image acquisition, autofocus, montaging, macros, tasks, tilt series, or any other extended sequence of actions.  It will generally stop the activity as soon as possible after the current action.

Macro/One Loop

If the Tilt Series Controller is not active, this button will execute the macro shown on the button; otherwise it will execute one loop of the tilt series.  The program will stop at the same place where the End button would stop it, after finishing all actions at a tilt angle.

Macro/One Step

If the Tilt Series Controller is not active, this button will execute the macro shown on the button; otherwise it will advance one step of the tilt series.  There are many more steps in the loop of the Tilt Series Controller than actual actions under particular conditions, so in many cases nothing will appear to happen when this button is pushed.

Macro/Back Up

If the Tilt Series Controller is not active, this button will execute the macro shown on the button; otherwise it will open the Tilt Series Back Up dialog box

Macro Selector Spin Buttons

These spin buttons will change the macro number activated by the associated Macro button.


Image Alignment & Focus Panel

Align to

This button will align the image in buffer A to the image in the indicated buffer, using cross-correlation.  The buffer holding the image being aligned to is called the autoalign buffer.  This buffer is determined as follows:

When not in low dose mode, if the Copy on Save button in the Buffer Control panel is selected, then images in A will be aligned to the buffer where images are copied when saved to file.  If the button is not selected, then B is the autoalign buffer.

When in low dose mode, there are actually two autoalign buffers: the first for images from the Record area, the second for images from the Trial or Focus area.  If the Copy on Save button is selected, then Record images will be aligned to the buffer where saved images are copied, while Trial or Focus images will be aligned to the next buffer.  If the button is not selected, then the two autoalign buffers are B and C.

Autofocus

This button will run the autofocus routine to measure specimen defocus (based on the amount of image displacement when the beam is tilted) then change the defocus to your chosen target value.

Clear Alignment

This button will set the alignment shift of the image in the currently displayed buffer to zero.  If this is buffer A, then microscope image shift will also be changed by an amount corresponding to the image alignment shift.  Thus, this button can be used to undo a bad autoalignment.

Reset Image Shift

This button will reset the microscope image shift to zero and move the stage to compensate for the change in image shift.  If the specimen is tilted, defocus will also be changed to compensate for the change in Z height of the feature in the center of the field.

Because of inaccuracies in stage movement, image features can end up 0.5 micron or more from their original positions.  This button is useful for a quick reset when you are still moving around on the specimen.  If you want to keep a feature of interest centered through the reset, then select the Reset & Realign command  in the Tasks menu.

Move Stage for Big Mouse Shifts

When this check box is selected, moving the image in Buffer A with the right mouse button will result in the stage being moved by the corresponding amount, provided that the move is bigger than a threshold that can be adjusted with the ‘Set Threshold Shift’ button.  Moves less than the threshold amount will cause the usual image shift instead.  If you hold down the Shift key while shifting with the right mouse button, the stage will be moved regardless of the size of the move.

Set Threshold Shift

Use this button to set the thresholds for moving the stage instead of shifting the image with the right mouse button.  First you will be asked to enter a relative threshold, expressed as a fraction of the size of the camera field.  Then you will enter an absolute threshold in microns.

Center Image Shift on Tilt Axis

When this check box is selected, SerialEM will move the origin for image shift laterally from the optic axis to the tilt axis.  Resetting image shift will then set the image shift to a position on the tilt axis, and the IS readout in the Microscope Status panel will be 0.0 even though image shift is nonzero (as can be seen in the Tecnai interface).  With this option enabled, lateral movement and focus changes will be minimized during a tilt series.  However, turning the option on or off will misalign the objective aperture on the 300 KV Tecnai.  When the option is selected, the program will move the image shift origin when it is started and reset the origin when it exits.

Trim Dark Borders in Autoalign

When this check box is selected, SerialEM will always analyze each image being correlated in an autoalign and exclude dark regions that appear to be outside the area of the beam.  Ordinarily this analysis is done only in low dose mode and when performing tasks at lower magnification.

Assess Multiple Peaks in Autoalign

When this check box is selected, the autoalignment procedure will evaluate multiple peaks in the crosscorrelation.  Each peak in the correlation corresponds to an alignment shift between the images in question.  For each peak, the procedure will correlate relatively small areas centered in the overlap area between the images with the given alignment shift.  The peak that gives the highest local correlation will be selected.  This option is useful if a periodic specimen (e.g., calibration grating) is being aligned.  For aperiodic specimens, it can cause alignments to fail and should not be selected.

Set Autoalign Trim Fraction

Use this button to set the fraction to trim off each edge of the images that are being aligned.  For example, with a fraction of 0.1, 100 pixels will be trimmed off each side of a 1K x 1K image.  In rare cases, the default value of 0.04 will not be adequate and a larger fraction is needed to exclude peripheral parts of the image with strong contrast.  Because this is needed so rarely, this value is not stored in the settings file between SerialEM sessions.


Low Dose Control Panel

Low Dose Mode

This check box turns low dose mode on or off.  When low dose is turned on, the current area is undefined unless the screen is down.  When low dose is turned off, the program returns to the Record area.

Electron dose of current area

If electron dose is calibrated, the dose will appear for the current area in the upper right corner of the Low Dose panel.  This dose will be updated continuously when the “Continuous update” option is selected and will also we changed when the exposure time changes for the current area.

Mag & beam settings of current area

The line will show the name of the current area and its magnification, spot size, and brightness setting as given by the C2 condenser lens strength.  The line is shown in black when the ‘Continuous update’ option is selected, to signify that the values can be readily changed.  Otherwise the line will be grayed out and be hard to read.

Continuous update of mag & beam

When this option is selected, any change of magnification, spot size, or brightness via microscope controls will immediately change the parameters of the current area.  Otherwise, such changes will be lost when a new image is acquired from the given area.

Area to show when screen down

These radio buttons allow you to select which area is shown when the screen is down.  The area shown becomes the current area when the screen is down.

BLANK BEAM when screen down

This option will prevent specimen exposure while the screen is down.  It could be useful for making an area be the current area without exposing the specimen.  You might want to do this to view or adjust its parameters.

Unblank button and blanked indicator

The Unblank button can be used to unblank the beam until the next image acquisition, which will restore blanking when it is done.  (Other actions may also turn the blanking back on.)  The principal use of this button is to unblank the beam before aligning an energy filter.  To the right of the button, the text “Blanked” will appear whenever the beam is blanked, even transiently during exposures when not in low dose mode.

Normalize beam through View

When this option is selected, changes in intensity and spot size when going between areas will be normalized by passing through the View area intensity and spot size.  This normalization is intended to compensate for the lens hysteresis that makes the beam position depend on the recent history of intensity changes, a pronounced effect on the JEOL scope.  With the option on, the beam position in the Record area, for example, should be the same regardless of whether the program has just come from the View area or has been going back and forth between the Record and Trial/Focus areas.  If different areas still have different beam positions, use the option for setting ‘Additional beam shift’ for some areas.  It may be necessary to set the property LowDoseBeamNormDelay to a higher value for maximum effect.

Keep Focus and Trial identical

When this option is selected, the parameters of the Focus and Trial areas will be kept the same in all respects.  It is desirable for these areas to be in the same location because of the large settling time that would be required after moving the beam from one side of the Record area to the other, and because of the limited space inside the objective aperture on the 300 KV Tecnai.  You might want different brightness settings, in which case you would have this option selected while positioning the areas, then unselected to adjust the brightness of one.

Copy current area mag & beam to

These buttons will copy the exposure parameters (but not the location) of the current area to one of the 4 areas: View, Focus, Trial, or Record.  This would be useful mostly when initially setting up the areas.

Center Unshifted

When you push this button, the View and Record areas will be set to have no image shift relative to the center of the viewing area, and the Focus and Trial areas will be displaced by the full amount of their image shifts from the center of the viewing area.  In other words, when the overall image shift (as reported in the Scope Status panel) is reset to zero, the Record area will be at a true image shift of zero.

Balance Shifts

When you push this button, the image shift of the Focus and Trial areas will be divided between those areas and the Record area.  The Record will be displaced to one side of the center of the viewing area, and the Focus and trial areas to the other side (assuming they are on the same side).  This means that when image shift is reset to zero, there will actually be substantial image shift when viewing the Record area.

Define position of area

Select Focus or Trial to adjust the position of these areas relative to the Record area.  (If ‘Keep Focus and Trial identical’ is selected, it does not matter which area is selected here.)  With one of these options selected, SerialEM will try to display a marker point on the image in buffer A showing the center of the area being defined.  You can adjust the position of the area in 4 ways:

1) by clicking with the left mouse button in a View image or in an image from the area being defined;

2) by entering a positive or negative distance in the ‘Position on tilt axis’ text box;

3) by shifting an image of the area being defined with the right mouse button (although shifting a Record or View will still change image shift as usual);

4) and even by adjusting image shift through microscope controls when the screen is down.

When you start defining an area, the area that will be shown when the screen is down will be changed to the area being defined.  The previous area to show will be restored after the you stop defining the area.

You should select ‘None’ after adjusting the area to avoid inadvertent changes, and to restore the expected behavior when shifting an image with the right mouse button.  Note that shifting the area being defined would change the

Position on Tilt Axis

When the position of the Focus or Trial area is being defined, this text box will show the center-to-center separation between that area and the Record area.  You can enter a number to set this distance as desired.

Area separation

When the position of an area is being defined, this line shows the minimum separation between that area and the Record area.  This separation is the distance between the edge of a circle drawn just around the outside of the area being defined, as defined by its camera parameters, and the edge of the Record area as defined by its camera parameters.  If ‘Keep Focus and Trial identical’ is selected, the larger of the two areas will be used for this computation.

Additional beam shift

The controls in this box allow you to impose an independent beam shift for each area.  You can use this is correct for imperfections in beam centering between the different areas, such as due to mag changes are inadequacy in the coupling between beam shift and image shift.  You can also use it to shift the beam in the trial and focus areas farther away from the record area than it would be if it were centered.  You should keep one area such as the Record area as a reference area in which the beam is not shifted.  To set beam shift for an area, first be sure that the beam is centered in the reference area, then go to the area of interest, turn on the ‘Set’ checkbox, move the beam to the desired position and turn off ‘Set’.  At this point, if beam shift is calibrated, the value of the shift will appear next to the ‘Reset’ button.  This shift is in microns and in specimen coordinates, where X is along the tilt axis.  Press the ‘Reset’ button to return the shift of the current area to zero.


Montage Control Panel

This panel contains options for controlling montage acquisition.  It opens when montaging is initiated and closes when a montaged file is closed.

Start

The button will start a montage acquisition.

Prescan

This button will start the relatively fast acquisition of binned down images that are used just to compose an overview of the region being montaged.

Current Z

The value shown in the text box is the Z or section number that will be used for the next montage acquisition.  You can enter a new number or use the up or down arrows to increase or decrease the value by one.  If you set the value to a section number that has already been acquired, you will overwrite the stored data for that section, which the program will ask you to confirm.  If you increase the value you may leave a gap in Z values.

Bin Overview

Ordinarily, an overview image will be subsampled so as to make the overview size comparable to the size of a single frame image.  This subsampling is referred to (inaccurately) as binning.  You can adjust this binning to a lower number to see more detail when you zoom up the overview image.  When the Navigator sets up a montage, the overview binning will be set to 1, under the assumption that you will want to see all the detail in a map montage.

Bin Prescan

By default, a prescan montage is taken at as high a binning as possible to take the least amount of time.  You can set this binning to a lower value if you want to see more detail in the prescan.  Note that the overview binning is always 1 for a prescan montage, so you need not adjust that binning as well.

Correct Drift

If this option is selected, the program will use the misregistration between a pair of successive pieces to predict and correct for the error in position of the next acquired piece.  When there is significant drift, this option can reduce the mean error before shifting pieces into registration, and it can prevent very large errors between successive columns of pieces.

Change Focus with Height

If this option is selected, focus will be changed for every piece that is acquired from a tilted specimen.  This focus change will compensate for the differences in Z height at the centers of the pieces.

Show Overview at End

If this option is selected, the overview image in buffer B will be shown at the end of every montage acquisition.

Align Pieces in Overview

If this option is selected, each piece will be shifted into alignment with preceding pieces as it is added to the overview image, based on the shifts in the overlap zones that it shares with preceding pieces.  This may give a seamless overview, or it may not if there are errors in measuring shifts in the overlap zones.

Treat as Very Sloppy Montage

If this option is selected, SerialEM will analyze the overlap zones with the same parameters that are invoked by default with the VerySloppyMontage option in Blendmont.  The large correlation areas and other parameter settings will give much better results with montages where the errors are large and where there may be strong edge effects from the edge of a cell or a grid bar.  The option is turned on or off depending on whether you choose to montage by moving the stage in the Montage Setup dialog box , but you can then toggle it as desired when reading in a montage from a file.  This option is not the default for two reasons: there is significantly more computation time with the larger correlations, which will be noticeable when reading from a file; and there is very little experience with using these parameters for normal montages based on image shift that have less error.


Filter Control Panel

This panel contains options for controlling the Gatan Imaging Filter (GIF) and the magnification and intensity settings of the Tecnai when operating the GIF.  On the JEOL with omega filter, the options related to lens modes, camera selection, pixel size, and intensity are not needed and will not appear.  The energy shift on the JEOL is referred to as spectrum offset here.

EFTEM Mode

The button will toggle EFTEM mode on and off.  When EFTEM mode is turned on and the screen is up, the microscope will be placed in one of the EFTEM magnifications.  If the screen is down, the microscope will stay at a regular magnification if the ‘Regular mag with screen down’ option is selected

Refine ZLP

This button will start a procedure for refining the alignment of the zero-loss peak (ZLP).  The procedure will use a slit of 20 eV, start at the current alignment, and take pictures at progressively lower energies in 2 eV steps until the edge of the slit is moved past the ZLP.  It will generally work only if the ZLP is already roughly aligned (within about 15 eV).  If it fails to detect the peak, it will try additional iterations with the starting position shifted left or right by the slot width (except in Low Dose mode).  Pictures are taken with the Trial parameters in normal mode or with the Preview parameters in Low Dose mode, except that the routine will try to lower the exposure by a factor of 10 and double the binning.  A small existing energy loss setting in the filter parameters will be taken as a starting position for the procedure and set to zero at the end.  This will occur automatically if the loss is less than half of the slit width setting in this panel; if the loss is more than half but still less than the slit width setting, the routine will ask whether you want to take the loss as a starting pointing.  The slit must be in for the loss to be treated in this way.  In Low Dose mode, the routine will look first at the properties of the current area, if it has the slit in, then examine the properties of the Record area.

Adjust Slit Offset     (JEOL with no negative energy shift)

If the filter allows only positive energy shifts, then several program features will not work unless the slit is centered with an energy loss sufficiently far from zero.  These include the procedure for refining the ZLP, the ability to apply magnification-dependent changes in the energy shift, and the procedure for calibrating the mag-dependent shifts.  This button will facilitate adjusting the slit position so that it is centered on the ZLP with a positive energy offset.  After you press it, enter the desired offset and press OK.  The filter will be placed into spectroscopy mode with the slit in, allowing you to center the slit on the energy-shifted peak.  After you make this adjustment, press OK.  You should then be able to run the Refine ZLP routine to get the ZLP aligned as well as it would be on a GIF.

Regular mag with screen down

This button determines whether you see an image at normal magnification or in the EFTEM reduced magnification when the screen is down.  The normal magnification provided by this option is almost always preferable.  However, if you want to check the size or centering of the beam in EFTEM mode, or align the crossover correction at lower magnification, you would turn this option off.

Filter (slit in)

Select this option to insert the slit and acquire energy-filtered images.  The check box will also reflect the current state of the slit when other programs modify it.  When the slit is inserted, the specified energy loss (either zero or the amount in the ‘Loss’ text box), plus the mag-dependent energy shift, if any, is used to set the filter’s spectrum offset.  If the slit is not inserted, then spectrum offset is maintained at zero.

Slit width

Enter a width in electron volts into the text box, or use the spin buttons to increase or decrease the width by 5 eV.  The slit is set to this width regardless of whether it is inserted or not.

Zero loss

Once filtered imaging is selected, you can filter at either zero loss or a specific energy loss.  This check box allows you switch easily between the two.  With the box checked, images will be taken of the zero loss peak.  With the box unchecked, images will be taken at the energy loss specified in the ‘Loss’ text box.

Loss

For filtered imaging, enter an energy loss in electron volts into the text box, or use the spin buttons to increase or decrease the loss by 1 eV.

Adjustment readout

This text shows the energy shift that will be applied to compensate for magnification, slit width, and ZLP alignment changes if the ‘Do mag-dependent energy shifts’ option is selected.  The value is set to zero when SerialEM detects that you have aligned the zero loss peak through TUI, it changes by the calibrated amount of mag-dependent energy shift when you change magnification, it changes by half of any change in slit width that you make, and it changes by the amount determined by the Refine ZLP procedure.  On a JEOL, the adjustment will include the offset imposed to keep energy shifts positive.  The factors determining the adjustment value will be retained for up to 1 hour between sessions of the program, so you should be able to exit and restart without needing to realign the ZLP.

Net offset readout

This text shows the spectrum offset currently being applied.  It should be zero when the slit is not inserted, and the sum of your selected energy loss and the adjustments being applied when the slit is inserted.  It should match the value for spectrum offset in the Tecnai EFTEM panel and is intended to reduce confusion when you see a non-zero number over there.

Match pixel size

Select this option to have the program adjust the magnification to minimize the change in pixel size when switching between EFTEM and regular magnifications.  If the option is not selected, you can get some rather large changes in magnification because the EFTEM and regular lens programs do not match up very well.  This feature is not active in low dose mode, where magnifications are defined as part of each exposure area.

Match intensity

Select this option to have the program change beam intensity when switching between regular and EFTEM magnifications, so as to maintain a constant electron flux per camera pixel.  With this option active, you can set the beam to give a particular screen current with the screen down, and get a predictable exposure level when you raise the screen.  This option and ‘Match pixel size’ can be selected independently, and will operate independently.  This feature is disabled in low dose mode because intensities are defined as part of each exposure area.

Do mag-dependent energy shifts

If this option is selected, the program will adjust spectrum offset to keep the slit centered on a desired energy loss when magnification or slit width is changed.  In order for this to work correctly, mag-dependent energy shifts need to be calibrated and you need to be sure to inform SerialEM whenever you align the ZLP.

Switch cameras automatically

With this option selected, the program will automatically select the GIF or upper camera when EFTEM mode is turned on or off, and it turns EFTEM mode on or off depending on which camera is selected.  To protect the upper camera from the intense beam in EFTEM mode, this option is enabled only in Administrator mode.


Camera Setup Dialog

This dialog box allows you to specify exposure parameters for image acquisition.

Camera radio buttons

If you have multiple cameras, each available camera will be listed here.  Each camera has an independent set of parameters.  The current camera will be the one selected.  If you select a different camera, that one will become the current camera when you press OK in this dialog, but not if you press Cancel.

Parameter set radio buttons

There are five independent parameter sets for taking pictures.  Use these buttons to select a set and view and adjust its parameters.  Whenever you change from one set to another, the parameters are stored for the set that you are leaving.

Copy from other camera

This button is available if there are multiple cameras.  Press it to copy parameters for the current parameter set from the camera listed on the button.  Acquisition area will be scaled appropriately if the two cameras are different sizes.

Acquisition

These buttons determine whether a single image will be acquired or whether acquisition will repeat automatically.  Continuous acquisition is relatively slow in SerialEM and thus not much use.  If you do start a continuous acquisition, you can stop it by pressing the space bar or Ctrl H, or with the Halt command in the Camera menu.

Processing

These buttons determine whether you will acquire an unprocessed, raw image from the camera, an image with the dark reference subtracted, or a gain-normalized image.  An image is gain-normalized by subtracting a dark reference then dividing by the gain reference prepared in Digital Micrograph.

Remove X-rays

This checkbox will activate removal of X-rays from an image taken with this parameter set.  It will appear only if the ShowRemoveXRaysBox property is set for the particular camera, or if one of your parameter sets already has the option checked.  Use this option only if necessary to prevent bad tracking due to large X-rays, since the X-ray removal in SerialEM is not as effective or reliable as the removal with Ccderaser in IMOD.  The parameters controlling the X ray removal are adjusted through the Set Image Criteria command in the Processing menu.

Binning

These radio buttons present the options for binning an image directly as it is read out of the CCD camera.

Exposure time

This text box allows you to enter the exposure time in seconds.  Unlike in DM, exposure time will not change when you change binning.

Drift settling

This text box allows you to enter the amount of time that the specimen will be exposed to the beam before the picture begins being recorded on the CCD camera.  This will minimize the effects of image drift when the beam first hits the specimen.

Shutter mode

These radio buttons allow a choice of how shutters will be operated to expose the specimen and camera.  The first two options correspond to the two shuttering modes available in DM:

1) Beam blanking only uses the shutter above the specimen; the beam is on the specimen only during the

exposure time, unless drift settling is non-zero.

2) Film shutter with beam blanking uses a combination of shutters below and above the specimen.  If drift settling is zero, the specimen is exposed for a fixed amount before image acquisition, while the CCD is cleared.  The ‘Minimum time if not 0.0’ listed below the drift settling box is based on this clearing time.  Unfortunately, the specimen is also exposed throughout the readout of the camera.

3) Dual shuttering for minimum exposure is accomplished by SerialEM using the beam blanker above the specimen to eliminate the unwanted exposure in DM’s film shutter option.  (This blanker is separate from the beam shutter that DM uses.)  SerialEM can thus provide a full range of drift settling times, both longer and shorter than the clearing time, without the undesirable effects that occur with drift settling through DM.  Exposure is also eliminated during camera readout.  When drift settling is zero, SerialEM simply uses DM’s beam shutter mode.  As long as this shuttering mode is working reliably, there is no reason not to have it selected for all parameter sets.

For a Tietz camera, only two shutter modes are shown.  In the first mode, the specimen will be exposed only during the drift settling time, if this is possible, plus the exposure time.  In the second mode, the beam will be left on the specimen continuously.

Force new dark reference next time only

If this option is selected, SerialEM will force DM to take a new dark reference the next time a picture is taken with this parameter set, and then the option will be cleared.

Take new dark reference each time

If this option is selected, SerialEM will force DM to take a new dark reference each time a picture is taken with this parameter set, which should be unnecessary unless DM’s drift settling is used in an older version of DM.

Average dark reference

If this option is selected, then the dark reference will be averaged the number of times indicated in the text box.  Averaging can be helpful for low-exposure images if the camera readout is particularly noisy. This selection, like everything else on the dialog, is specific to the camera and parameter set.  Checking this box does not affect when a dark reference is taken; if you change the setting, use “Force new dark reference next time” to get a new dark reference.  When an averaged dark reference is taken, the status bar will display “Dark Ref #n” instead of “Getting Dark Reference.  If you press “Stop” before the dark reference is finished, a dark reference will be saved based on the average of the references already obtained, and will be used on the next acquisition.

Top – Left – Bottom – Right

These text boxes display and allow you to change the coordinates of the area to be acquired.  As in DM, Y coordinates start with zero at the top.  Unlike in DM, coordinates are always expressed in terms of the full, unbinned size of the CCD camera.

Up-down and Left-right Spin Buttons and Recenter Button

The two pairs of spin buttons allow you to shift the capture area in any desired direction without changing its size.  The Recenter button will undo these shifts and center the area on the camera.

Quarter – Half – Full – Wide Quarter – Wide Half

These buttons provide the most convenient way to select the area of the camera to be acquired.  The quarter and half buttons refer to linear dimensions.  The wide quarter is one quarter of the height and one-half of the width of the camera, while the wide half is one half the height and the full width of the camera.  These options are provided because camera readout time is dominated by the number of rows being read out, and a rectangle gives more pixels of data per unit time than a square.

10% Less – 10% More – A Bit Less

These buttons provide a convenient way to adjust the area being acquired to sizes other than the stock ones provided by the buttons above.  Again, the changes are in linear dimensions, not areas.  Because of rounding, the ‘10% Less’ button will not exactly undo the change made by the ‘10% More’ button, and vice versa.  To get back to a regular size, just push one of the upper buttons.

The ‘A Bit Less’ button is useful for creating parameter sets with a slightly different sizes so that they will have independent dark references in DM.  If you have two parameter sets with the same image size and binning but different exposures, DM will take a new dark reference every time you switch from one to the other.  Making one set ‘a bit less’ in size will prevent this.

Dose readout and Update Dose button

If electron dose is calibrated for the current spot size and intensity, the estimated dose will be shown for the given exposure.  This readout is refreshed whenever you change parameter sets or when you press the Update Dose button, so you can change brightness and use this button to see the effect of the change.

OK – Cancel

When you press the OK button, any changes in the current parameter set will be saved; if you press Cancel, such changes will be discarded.  Note that changes are saved for a parameter set whenever you switch to another set.


Gain Reference Dialog

This dialog box allows you to acquire a gain reference for the current camera.  This reference is separate from the gain reference that DigitalMicrograph maintains, and once a reference has been obtained through SerialEM, SerialEM will use this reference instead of DigitalMicrograph’s reference.  There are two advantages to using a SerialEM gain reference:

1)	If SerialEM’s capability for removing X-rays from dark references is configured, the gain reference will be protected from the effects of X-rays in its dark reference (which produce hot pixels in the gain reference.)

2)	If you have a 4K camera with a slow readout and only need to use it as a 2K camera (e.g., fiber optic camera at 300 KV),  SerialEM allows you take just a gain reference binned by 2, which is much faster than taking a reference in DigitalMicrograph.

To acquire a gain reference, you want to have a fairly bright, uniform beam with no specimen.  The beam should be spread larger than the field of the camera, and will be more uniform if it is spread as large as the whole viewing screen.  Check the following parameters and press YES to proceed.

Target number of counts

SerialEM will find the exposure time that will give this number of counts in each image that it takes.  Enter a value in the text box or use the spin buttons to change the value by 500 .  A high value (e.g. 8000) is generally appropriate for a sensitive camera, even if you are going to take low-dose images.  However, if the camera is less sensitive, it may be necessary to make the target about the same as the number of counts expected in a typical image with normal exposure.

Number of frames to average

Use this entry to set the number of images to acquire and average.  Five to ten frames will usually give good results; the higher number may be needed to average out X-rays more.

Reference Binning

Select 1 to make an unbinned gain reference, which can be used to normalize images with any binning but could take a long time to acquire; or 2 to make a gain reference binned by 2.  The latter can be used only to normalize images with even binnings.  The text on this radio button will say whether an image with odd binning will be normalized with an older unbinned reference from SerialEM or with a reference from DigitalMicrograph.

Average dark reference

Select this option to have the dark reference used for the gain reference images averaged by the number of times indicated in the text box.  This averaging will reduce the effects of X-rays in the dark reference if you do not have X-ray removal set up or if it is not working well with the particular kind of X-ray signals produced in the camera.

Calibrate dose with last image

If this option is selected, then the program will use the last reference image to calibrate electron dose for the current spot size (see Electron Dose Calibration ).  In order for this to be as accurate as possible, the spot size will be normalized after you press the Yes button.  This may change the beam position and brightness, so there will then be a message box reminding you to check the size and centering of the beam.  If this is too annoying and you really do not care about dose, then deselect this option.


Montage Setup Dialog

This dialog box allows you to set the parameters for a montage, see the total area to be acquired, and view the parameters after montaging has been initiated.

Camera radio buttons

If you have multiple cameras, each available camera will be listed here so that you can specify or see what camera will be used for montaging.  The current camera will be the one initially selected.  If you select a different camera, it will become the current camera when a montage is started.

Magnification

This setting is initially based on the current microscope magnification, but you can change the magnification if desired using the spin buttons.  SerialEM will automatically switch to this magnification whenever acquiring a montage.  You can also change the magnification after montaging has been started, but you will have to confirm this change the first time you do so.

Binning

This setting is initially based on the current value in the Record parameter set, but you can change the binning when setting up the montage.  If you do so, the program will adjust the exposure time by the square of the change in binning, but you will also be asked if you want to adjust the exposure and drift settling parameters yourself.  If you have already started montaging, you may be able to change the binning if it is compatible with the size of the pieces.

Pixel size

This value is based upon the magnification, the binning, and the calibrations in SerialEM.

Number of pieces in X and Y

You can set the number of frames of the montage in the X and Y directions by entering numbers in the text boxes or using the spin buttons.

Piece size in X and Y

You can set the size of each piece in X and Y by entering values in these text boxes.  The default is the full size of the CCD camera.

Overlap in X and Y

You can set the amount of overlap between pieces by entering values in these text boxes.  The default is 10% of the frame size of the CCD camera.  These values may be changed when new piece sizes are entered.

Move Stage instead of Shifting Image

Select this option to have the montage acquired by moving the staging instead of using electronic image shift.  Image shift is quite accurate but has a limited range at medium and high magnifications (less than ~15 microns at 200 KV, less than ~3 microns depending upon objective aperture size at 300 KV).  Stage shift can be used when these limits need to be exceeded.  Note that in LM mode, image shift has a much higher range, so you may be able to use image shift instead of stage shift at the highest LM mode magnifications.

Do Full Rectangle; Ignore List of Pieces to Skip

When a montage is set up by the Navigator, it will compose a list of pieces that do not need to be acquired because they fall outside the polygon used to define the montage area.  The option allows one to acquire the complete array of pieces, including ones on the list to skip.

Total area and Update

The total area is computed based on the pixel size, number of pieces, piece size and overlaps.  It is recomputed whenever you push a button or leave a text box, but to be sure the value is current, just push the Update button.


Macro Control Dialog

This dialog box allows you to specify conditions for stopping a macro from looping.  Each limiting condition is activated by a check box; once it is active, you can enter a limiting value in the associated text box.

Stop if tilting up above or down below

These two entries will set the limits for tilting in the positive or negative directions, respectively.  The program will stop the macro after executing a step that tilts beyond the given limit.

Stop if image level is below

This entry will cause the macro to stop if images are too dark.  The program will measure the mean counts of the image in buffer A after executing a command that acquires a single image or a montage or autofocuses.

Stop if montage error exceeds

This entry will be compared to the mean displacement error after a montage acquisition, and the macro will stop if the error is too high.

Limit number of macro runs to

This entry can be used to limit the total number of times that a macro will repeat automatically.

Stop if image shift exceeds

This entry will cause the macro to stop whenever image shift exceeds the given value.


Tilt Series Setup Dialog

This complex dialog box allows you to set all of the parameters for a tilt series.  Some of the parameters that can be changed here can also be set through other menu or control panel options.  When you first open this dialog, the program makes some assumptions to set some initial values, but you are able to change all values.  If you have already started a tilt series, many of the options will be disabled.  For an overview and a list of steps commonly used in starting a tilt series, see the section on Acquiring Tilt Series.

The entries are organized into the following groups:

Camera Selection	Select the camera for the tilt series
Tilt Angle Specifications	Control the starting and ending angles and angular increment, and the delay time after tilting
Size and Image Shift Specifications	Control magnification, image shift resetting, and zero-loss peak alignment.
Beam Intensity Control	Control beam intensity changes.
Autofocus Control	Control autofocus behavior.
Initial Actions	Select options for startup phase of series.
Tracking Control Parameters	Set parameters to control prediction and tracking behavior.
Action Buttons	Actions upon closing the dialog.

Camera Selection

If you have multiple cameras, each available camera will be listed here so that you can specify or see what camera will be used for the series.  The current camera will be the one initially selected.  If you select a different camera, it will become the current camera when the series is started.

Tilt Angle Specifications

This grouping contains various controls related to tilting.  The increment, delay time, and choice of cosine tilting duplicate the entries that can be made through the Tilt Control panel .

Start at

Enter the starting tilt angle in this text box.  If you are already at high tilt when you first start the setup dialog, the box will show the current tilt angle; otherwise it will contain a default angle.  After a tilt series is started, this value cannot be changed.  The starting tilt angle can be positive or negative.

End at

Enter the ending tilt angle in this text box.  When you first start the setup dialog, this box will contain the negative of the value in the starting tilt angle text box.  The ending angle can be changed at any time.

Basic Increment

Enter the basic tilt increment in this text box.  If you have chosen to tilt with a fixed increment, this value will be that increment.  If you have chosen to have increments proportional to the cosine of the angle, this value will be the increment at zero tilt, and the increment will be progressively smaller at higher angles.

Increment proportional to cosine of angle

Select this option to have tilt increments proportional to the cosine of the tilt angle.  The increment would have its basic value at zero tilt, half of that at 60 degrees, and one-third of it at 70 degrees.

Total # of tilts

This status message will show the total number tilts for the given starting and ending angles, increment, and choice of regular or cosine tilting.  If you make a change in one of the text boxes, this message will be updated when you leave the text box.

Delay time after tilting by basic increment

Enter a delay time in seconds in this text box.  This delay will be applied before taking an image, but only under certain circumstances, i.e, when taking an image with the Record parameter set, when autofocusing, and when finding eucentricity.

Size and Image Shift Specifications

Magnification

Use the up and down arrows to change the magnification at which the tilt series will be obtained.  When you first start the setup dialog, the current microscope magnification will be shown.  If you change microscope magnification after starting a tilt series (or indeed, after leaving this dialog box with a magnification value set), the Tilt Series Controller will automatically restore the magnification when it resumes operation.  If you are montaging, the magnification cannot be changed through this dialog box, only through the Montage Setup dialog box .

Binning

This control shows the current binning of the Record parameter set.  You can use the up and down arrows to change the binning (unless you are montaging).  If you do change the binning, the exposure time will be changed accordingly, but you will be offered the chance to check exposure and drift settings before starting the tilt series.

Pixel size

The status message shows the pixel size, given the magnification and binning.

Track at lower magnification when needed

Select this option to have tracking images taken at a lower magnification at the start of the tilt series, or at other times when the Tilt Series Controller thinks that tracking at the regular magnification might fail, such as after resetting image shift.  With the option selected, you can adjust the magnification for the tracking images with the up and down arrows.  Choose a magnification where tracking occurs reliably.

Limit image shift

Enter the maximum image shift that should be allowed to accumulate before the program resets image shifts.

Refine zero-loss peak

If you have an energy filter, this option is available to refine the alignment of the zero-loss peak periodically during the tilt series.  After selecting the option, enter the interval in minutes between refinements.  The refinement will be done with a series of highly binned, low-exposure pictures.  In low-dose mode, the pictures are taken at the Record area and are based on the Preview parameters, but with even lower exposure if possible.

Beam Intensity Control

This grouping controls how beam intensity (brightness, condenser 2 setting) will be changed during the tilt series.  For a typical thick section, the transmitted intensity will decrease by a factor of 2-3 at the highest tilts unless the beam intensity is changed to compensate.

Keep beam intensity constant

Select this option if you do not want the program to change beam intensity during the series.  Unless you are in low dose mode, the program will actually do nothing to keep the intensity constant, and you are free to adjust it yourself.

Vary intensity as 1/cosine of angle to 1/n power

With a power of 1, this option would vary beam intensity proportional to the effective thickness of the sample as it tilts.  This would probably do a good job only if the fraction of electrons transmitted through the specimen and objective aperture is about 0.5 at zero tilt.  With a higher power, the variation in intensity will be less; for example, with a power of two the intensity will vary by the square root of the inverse cosine and thus change by a factor of 1.41 from 0 to 60º

Set intensity to keep mean counts constant

Select this option to have the beam continually adjusted during the series so that Record images will have the mean value specified in the text box.  Mean counts are measured in the tilt-foreshortened area of an image, i.e., the part that corresponds to what would be visible at zero tilt.  The line following this option will report what a Record image would have at the current intensity, based upon the counts in the image in Buffer A.

Taper counts down at high tilt

If you have selected the last option to keep mean counts constant, then this option is available to reduce the target mean value at high tilts.  This is useful if the beam cannot be condensed enough to give the desired number of counts without becoming smaller than the camera’s field of view at the highest tilts.  When you select it, you can enter a number of counts in the first text box and an angle in the second box.  Enter a positive angle; it will apply on both the positive and negative sides of the tilt series.  The target value will change linearly from its standard value at this angle down to the reduced value at the extreme angle of the tilt series.

Keep increase intensity below current value (use if intensity already set up)

If you are starting at high tilt and have already set up the intensity that you want at the starting tilt, use this option instead of specifying tapering of counts.  This option will maintain the intensity at the maximum amount until the target counts are reached at a lower tilt angle.

Do not increase intensity above value for first saved image

This option can be used to keep the beam from being condensed any more than it was at the beginning of the tilt series.  This would be useful if you end up going to higher tilts at the end of the series than at its start, and could also prevent over-condensing the beam during the range of angles where the intensity is tapered.  The limiting intensity is the intensity value set just after saving the first image of the tilt series and adjusting the beam to achieve the target number of counts.

Use current intensity at zero tilt (increase to start series)

This option is relevant and enabled if you have selected inverse cosine intensity changes and are starting at high tilt.  Select the option if the intensity was set up to be appropriate at zero tilt.  When the tilt series starts, intensity will be increased by the appropriate inverse cosine factor.

Close column valves at end of series

This option provides the final control of beam intensity by allowing you have the column valves closed at the end of the series.  If you open the valves again and take additional pictures one tilt at a time with the One Loop button, they will remain open.

Average Record dark references

This orphan checkbox is used to specify whether averaging should be used when getting the dark reference for Record images during the tilt series.  Its setting overrides the state of the checkbox for averaging Record dark references in the Camera Parameters dialog box, so if you only want the averaging to occur for the one or two dark references used during a tilt series, select this option and leave the option in the Camera Parameters dialog unchecked.  The dark reference will be averaged by the number of dark images specified for the Record parameter set int the Camera Parameters dialog.

Autofocus Control

Defocus target

The target defocus value for autofocusing will appear in this text box and a new value can be entered.

Beam tilt

The amount of beam tilt used for autofocusing will appear in this text box and a new value can be entered.

Autofocus offset

The defocus change that will be applied before autofocusing will appear in this text box and a new value can be entered.

Skip autofocus

In low dose mode, this option can be selected to skip autofocusing during the series, which can be useful if autofocusing is not reliable.  The initial autofocus during the startup of the tilt series will also be skipped.

Do autofocus at certain interval

The Tilt Series Controller will skip autofocusing when the prediction for focus seems reliable enough; however, it will always do an autofocus when the tilt interval since the last time it was done is longer than the value entered in this text box.

Focus every time at high tilt

If this option is selected, the program will do autofocus at every tilt angle above the value entered in the text box.  Enter a positive angle; it will apply on both the positive and negative sides of the tilt series.

Check autofocus and stop if inadequate

If this option is selected, the program will run a routine to check the accuracy of autofocus at high tilt, and stop the tilt series if the accuracy is not greater than the specified amount.  The routine measures defocus at the current focus, and plus and minus 5 microns from that point, then reports what fraction of the actual change can be measured.  If this fraction is less than the amount specified in the text box, then the tilt series is stopped and some recommendations are given for proceeding.

Initial Actions

The options in this group will be enabled as appropriate, depending on whether you are at zero tilt or at the starting tilt of the series.

Buffer A has image of desired area

Select this option if you have left an image in buffer A aligned on the desired area before opening the dialog box.  If this option is not selected, the Tilt Series Controller will take its first reference for alignment as soon as possible, but the specimen may have drifted away from the desired position by then.  Thus it is generally wise to ensure that there is a good reference before you start.  This option is relevant when starting either at zero tilt or at high tilt.

Refine eucentricity

Select this option to have the eucentricity refined before going up to high tilt.

Leave mid-tilt anchor

Select this option to have the ‘Walk Up’ procedure leave an ‘anchor’ image near the angle specified in the text box.  When the tilt series reaches that angle, the anchor will become the reference for alignment, and this will pull the series into alignment.

Use mid-tilt anchor

Select this option if you have taken an ‘anchor’ image at an intermediate tilt angle which you want the program to use for alignment when that angle is reached in the tilt series.  Use the up or down arrows to select the buffer in which you left the image.  If you obtained the anchor with the “Walk Up & Anchor” command in the Tasks menu, then it will be in the last image buffer, so this buffer is initially the default.

Tracking Control Parameters

This grouping contains various parameters that govern the Tilt Series Controller’s behavior with regard to predictions for positional and focus changes.  The typical user needs to pay attention to just the first three.

Repeat Record if certain percentage of field is lost

With this option selected, the program will repeat the acquisition of a Record image when its error in position is sufficiently high.  The error is expressed as the percentage of the field of interest missing from the image.  In computing this fraction, the program excludes areas far from the tilt axis that will not be needed for a reconstruction.  Thus, at middle to high tilts, the errors in the Y direction (across the tilt axis) can be relatively high without triggering a reshoot.  Errors in the X direction (along the tilt axis), or errors in either direction at low tilt, will be constrained to the percentage set in the text box.

There are several reasons why you might not select this option, or might enter a particularly high value: if you have no interest in what is contained in the periphery of the area being acquired; if you are working at a high magnification where errors are intrinsically higher; or if you are doing low dose work and cannot afford to repeat exposures.  Note that in low dose mode, the program will stop and ask for confirmation before redoing a Record image.

Stop when Autoalign gives a large shift

With this option selected, the program will examine all Autoalignments at the regular magnification and will stop the tilt series if the alignment shift exceeds the specified percentage of the size of the image being aligned.  This may prevent the program from going astray on a wildly incorrect alignment.  When the tilt series stops, you can revert to the previous specimen position by clearing the alignment using the button on the Image Alignment and Focus control panel.  If the large image shift is correct, you can resume the tilt series instead.

Note that when montaging, the image size for this comparison will be that of the Montage Center, i.e., the size of a single frame, not of the whole montage.

Get tracking image when prediction error is high

This entry determines when the program will acquire a tracking image rather than relying on the prediction of the position on the next tilt.  The criterion that is entered is expressed as a percentage of the size of the camera area that is being recorded.  You may want to enter a larger value when working at high magnifications where errors are inherently larger.  On every tilt, the program computes how far the Record image on the previous tilt was from its predicted position in X and in Y.  It will choose to do tracking if the prediction error in either the X or the Y direction was greater than the criterion.  In addition, it computes a predicted position at the current tilt and the standard error of the prediction in both X and Y.  Tracking will occur if the square root of the sum of the squares of these two standard errors is higher than the criterion.

Track: Before – After – Before & after Autofocusing

These radio buttons will control where tracking will occur in the sequence of operations at each tilt.  The default is tracking before autofocusing, which will guarantee that focusing is done at the center of the field.  However, when drift is bad, the Record image will then be displaced because of the time spent focusing.  In this case, selecting tracking after autofocusing will keep the Record image from being off by much.  When tracking occurs after autofocusing, SerialEM will adjust the focus as necessary to compensate for the lateral shift in position.  If drift is really bad, one could select tracking both before and after focusing.

Stop after aligning tracking image

Selecting this option activates a manual tracking mode in which the program will stop after acquiring and aligning a tracking image.  You can then adjust the alignment position manually and resume.  When you resume with the One Loop button, the image in buffer A will automatically be taken as a new alignment reference.  Thus, in this mode one can simply adjust the tracking position as necessary and push One Loop to go on.  When manual tracking is no longer needed, turn off this option.

Align only with tracking images

This option is available in low dose mode, where the program will ordinarily align with tracking images in the Trial area when necessary, and refine the alignment on every tilt with images from the Record area.  The alignments occurring in these two separate areas can diverge from each other.  If this divergence is too much of a problem, this option can be used to disable alignment of the Record images.  It will cause a Trial image to be taken on every tilt and used for tracking.

Align with Preview before getting new track reference

In low dose mode, it is sometimes necessary to take a new tracking reference after a Record image.  If there is drift and time for acquiring the Record image is long, this reference will be in the wrong place.  With this option selected, the program will always take a Preview image and align it to the existing Record reference just before acquiring a new tracking reference.  Since the Preview should take less time to acquire, the track reference location will be more accurate.  This presumes that Preview is set up to take a fast, binned image that will not contribute much to the dose on the Record area.

Get new track reference if Record alignment differs

The criterion entered in this text box comes into play both in low dose mode and when low magnification tracking is enabled.  In both of these cases, it is possible for the alignment specified by the tracking images to diverge from the alignment of the Record images.  When the program takes a tracking image and uses it for alignment, then finds that the position of the next Record image is in error by more than this criterion, it will take a tracking image after the Record on the next tilt and use this image as a new reference.  This criterion should be set rather high, because minor misalignments are handled automatically by simply shifting the existing reference.

Repeat autofocus if error is high

When this option is selected, the program will repeat the autofocus procedure when the defocus measured by a first autofocus differs from the predicted value by more than the criterion entered in the text box.  It does this even if the prediction had a high standard error and was likely to be in error itself.

Do autofocus when prediction error is high

This entry determines when the program will autofocus rather than relying on the prediction of the focus on the next tilt.  This criterion is compared with two different errors.  At every tilt, the program computes a predicted defocus and a standard error for this prediction.  Autofocus will be done if twice this standard error is greater than the criterion.  In addition, if autofocus was done at the previous tilt, there is an actual error, the difference between the measured defocus and the predicted value.  Autofocus will be done if this actual error exceeded the criterion.

Action Buttons

GO

This button will start or resume a tilt series.  If a series was running and had been stopped, it will resume at the next logical step.

Single Loop

If the tilt series has not been started yet, using this button to start it will make it run through all of the preliminary steps before starting its regular actions at the first tilt angle.  This would then give you an opportunity to adjust specimen position or beam intensity. If a series was running and had been stopped, it will resume at the next logical step then go until all actions at one tilt angle are finished.

Postpone

This button will close the dialog box and save all parameter settings without starting or resuming the series.  It is useful when you realize that you need to take some preliminary action before starting the series, such as ensuring the centering of the image or adjusting beam intensity.  You could also use it if you need to change a control parameter during the tilt series and also need to resume at a step other than the current one.

Cancel

This button will close the dialog box and discard any parameter changes that you have made.


Tilt Series Resume Dialog

This dialog box allows you to resume a tilt series that has stopped, and to control the point at which it will be resumed.  It also lets you indicate that you have changed the image alignment.

Next action in sequence

The dialog box starts with a message describing what action will occur if you simply resume with the next action.  Certain events will make the program back up and repeat steps that have already occurred at the current tilt.  Such changes will already be reflected in this message.

Choose one option for continuing

This set of radio buttons presents the set of options for how the program will resume.  Inappropriate options will be disabled.  When montaging, the options will refer to taking a montage rather than taking a Record image.  Depending upon where you are in the tit series loop, some of the options may involve going back and repeating steps, whereas others may skip steps that you have managed by hand.  Any time that you select an option that will save a Record image, this image will overwrite any existing image at the current tilt angle.

1)	Resume with next action in sequence – This will be the action described in the message at the top of the dialog box

2)	Go on to next tilt – This option will not be available unless you have finished all actions at the current tilt angle.  It could be used to prevent the program from taking a new Record image and overwriting the one in the file.

3)	Save the Record image in Buffer A – Use this option if you have taken a new Record image that you want saved.

4)	Take new Record image and save it – Use this option to have a new Record image taken for you and saved to file.  The program will skip any scheduled tracking and focusing steps.

5)	Autofocus then take Record image – Use this option to start with the autofocusing step.

6)	Track, autofocus, and take Record image – Use this option to start with the tracking step.

7)	Low mag track, autofocus, and take Record image – If you have low magnification tracking enabled, this option is also available to let you resume with a low magnification tracking image.

Specify other conditions

Select the option ‘Use image in buffer A as reference for alignments’ if you have changed the alignment and taken an image at the new alignment.  The program will move this image into the autoalign buffer (D) to replace the existing reference.  In low dose mode, this new reference can be from either the Record area or the Trial area; the program will move it into the appropriate buffer (D or E) to replace the respective reference, and avoid using the image in the other alignment buffer for alignment.

Action Buttons

The GO button will resume te tilt series unconditionally; the One Loop button will make it stop after finishing all actions at one tilt; the One Step button will execute one step; and the Cancel button will just close the dialog box.


Tilt Series Back Up Dialog

This dialog box allows you to back up in the tilt series, assess the alignment or quality of images at previous tilts, and resume at a selected tilt angle.  When you do that, new images will be taken at each tilt and used to overwrite the existing images until you reach the place that you backed up from.  The dialog box presents a selection of tilts, listed by their Z values in the file and by their tilt angles.  Select one and push the GO button to back up to that tilt.

When you select an angle, the program will tilt back to that angle, compensating for backlash, read in the stored image for that tilt angle and place it in buffer A, and read in the stored image for the previous tilt angle and place it in buffer D (the autoalign buffer).  If you resume from that point, the program will start with tracking and focusing at the new tilt angle, aligning images to the reference from the previous angle.  If you are montaging, the program will recompose the center image for the selected angle and for the previous tilt.

If you tilt back too far, you can open this dialog box again and tilt forward again.  You can only tilt forward to the last angle that was finished, not necessarily to the angle at which you stopped.

If you are in low dose mode, the program maintains a stash of tracking reference images which it can access to set up buffers appropriately when backing up.  Specifically, it will place the stored image from the previous tilt in D and one of the tracking reference images in E.  You can then autoalign either Trial or Record images at the new tilt angle.  Your choice of angles to back up to may be constrained because of the limited number of tracking references available.

If you are working at a high magnification and need low mag tracking, you may have to get the specimen aligned manually after backing up, since there is not guaranteed to be an appropriate low mag reference in the second autoalign buffer.


Navigator Window

The Navigator window is used to keep track of stage positions and low magnification maps and can assist with locating positions on the grid after it has been rotated or repositioned in the holder.  Each stage position and associated information is referred to as an item. The window has four kinds of controls:

1)	At the top, properties of the currently selected item.

2)	Below item properties, some controls governing overall behavior.

3)	Below that, on the left, are pushbuttons for the most commonly used actions.

4)	In the lower right is a box containing a list of items and their properties.

The Navigator window can be resized to show more or less of the list box.

Label

The label is a short identifier that will be drawn on the image.  The Navigator will assign labels as sequential numbers but you are free to edit them to say something more meaningful.

Registration Point

Use this check box to mark a point as a registration point, which can be used to transform locations when the grid is repositioned in the holder.  The first time that you set up a registration, you can use the spin buttons to select a unique point number (this should happen automatically).  When you are adding corresponding points in a new registration, use the spin buttons to make each point number match the number of the corresponding point in the previous registration.

Corner Point

Use this check box to mark a point as a corner of an area for which you want to construct a montage.

Color

You can change the color of an item with this selector.  By default, single points are red, polygons are green, and maps are blue.

Draw

Use this check box to control whether an item is drawn on images that it falls within.

Acquire

Use this check box to mark an item as a position to be acquired automatically.  Once an image file is opened for montaging or saving of single frames, you can use the Acquire Areas command to acquire images at the sequence of locations and make each into a map.

Note

This text box allows you enter an arbitrarily long note about the current item.

Current Registration

The current registration starts at 1 and should be increased every time the grid is repositioned in the holder or the holder is replaced in the microscope. Use the spin button to change the current registration.  An item is associated with the current registration when it is created.  Images are also associated with the current registration when they are captured.  However, if you change the registration after acquiring an image and begin to draw points on it, the image’s registration will be changed to match the current registration, unless the image has already been defined as a map.

Draw All Registrations

Ordinarily the only items that will be drawn on an image are ones with the same registration as the image.  Use this check box to turn on drawing of items from other registrations.

Draw None

Use this check box to turn off drawing of all items.

Add Stage Pos

Use this button to add the current stage position as a point item.  Note that the stage position will be adjusted by any image shift, so it may not match the current X/Y coordinates of the stage.

Add Points

Use this button to start adding one or more points by clicking in an image with the left mouse button.  Each point will become a separate item.  Push the button again to stop adding points.  While you are adding points, you can press backspace to delete the points backwards one by one.

Add Polygon

Use this button to start drawing a polygon by clicking points in an image with the left mouse button. The polygon can then be used to define an area to be montaged.  Push the button again when you are done adding points; the last point will then appear connected to the first.  While you are adding points, you can press backspace to delete the points backwards one by one.

Move Item

Use this button to reposition the current item by clicking a new center position for it in an image.  Push the button again to stop moving the item.

Go To XY

Use this button to move the stage to the position of the current item in X and Y, leaving Z unchanged.  Image shift will be reset to zero when you move the stage.

Go To XYZ

Use this button to move the stage to the position of the current item in X, Y, and Z.

Go To Marker

The marker is the green cross that is deposited on an image by clicking with the left mouse button.  Use this button to move the stage to the location corresponding to the marker position in the currently active image.

Load Map

Use this button to load in the image for a map, if the current item is a map.  The image will be left in the buffer used for read-in images (typically E).  If the map is a montage, the overview will be copied from B to this buffer.  Maps can be loaded regardless of whether another file is open for saving images.

New Map

Use this button to define the current image as a map.  A map consists of an image that has been saved to a known file.  There must be an open file to create a map.  If it has single frames, the image in buffer A must have been saved to the file.  If the file is a montage, the image in Buffer B must be an overview from a montage capture.  It is possible to use an image or montage overview read in from a file to make a map, but this is discouraged because of the potential ambiguity about the stage position at which they were taken.

Delete Item

Use this button to delete an item from the list.  You will be asked to confirm deletion of a map because it would be hard to recreate.

List Box with Items

The list box shows all of the items and their properties.  It can be scrolled to bring text into view, or the whole window can be made bigger if desired.  The following actions can be done in the box:

•	Single clicking on an item will make it the current item.

•	Double clicking on a map will load and display the map.

•	Up and Down arrow keys will move the current selection up and down in the list, as long as the main program window has the keyboard focus.  Ordinarily these arrow keys would pan an image up or down, but when the Navigator is open they will move the list box selection instead.

•	Items can be reordered by drag and drop; click on an item and hold the left mouse button down while dragging the item to the desired place.

The properties shown for each item are the label, the color, the X, Y and Z coordinates, and the type of item (point, polygon, or map).  Next is the registration number with which the item is associated.  If the item is a registration point, the point number is shown as well, with an entry “#R#”, where the first # is the registration number with which the point is associated, and the second # is the unique point number for this point.  If the item is marked as a corner or an acquisition point, the letters C and A will appear, respectively.  Finally, the note will appear at the end of the line.


Macro Editing Window

These windows allow one to edit macros.  For a list of available commands see Macro Commands ; for general information about macros see About macros .  Several basic editing functions are available in the window.  Text can be selected with the mouse.  Text can be cut with Ctrl X, copied with Ctrl C, and pasted with Ctrl V.  Ctrl Z will undo the last action.  While you are editing a macro, the text in the window will not match the version of the macro stored in the program.  However, running the macro, closing the editing window with the OK button, or even exiting the program will transfer the text in the window to the program’s memory.

The buttons below the text area perform the following functions:

Run button - will save the macro in program memory and run it.

Cancel button - will close the window without saving the most recent changes in program memory.

OK button – will save the macro in program memory and close the window.

Load button - will allow you to read a macro from a file into the current macro.  Once a macro has been read from a file, the Save button will save it back to that file.

Save button - will allow you to save this macro to a text file.  If you have saved it to a file before, or if you loaded it from a file, it will be saved to the same file.  Otherwise you will enter the Save As dialog box to specify a file

Save As button- will allow you to save this macro to a different file via the Save As dialog box.


File Properties Dialog

This dialog box provides options for file storage.  Most of them have to do with how to treat unsigned 16-bit integer data, produced by 16-bit cameras.  According to the MRC standard, MRC files in integer mode store data as signed integers, ranging from -32768 to 32767.  However, 16-bit cameras have the potential to produce numbers higher than 32767.  SerialEM provides five (!) options for dealing with this possibility.

1)	Data can be saved in a non-standard unsigned integer mode (mode 6), which is supported in IMOD as of version 3.7 but may not be portable to other MRC file reading software.

2)	Data can be saved in a standard integer file with truncation at 32767.  If the counts in the images to be saved are not expected to be higher than 32767, this should not lose any data.

3)	Data can be divided by 2 when saved to file.  This will keep the data positive (and thus proportional to transmitted electrons through the specimen) but will lose one bit of precision.  Since the Gatan 16-bit cameras simply amplify the signal from the CCD chip 4 times as much as the 14-bit cameras, dividing by 2 probably just removes one bit of noise, which makes this an acceptable option for current US1000/US4000 cameras.

4)	Subtracting 32768 and storing with the standard file mode will preserve the camera values completely, at the cost of producing negative values in the output file.

5)	Data can be divided by 2 as soon as they are acquired, by selecting Divide 16-bit By 2 in the Camera menu.  They will thus never appear to be unsigned integers, making the options for treatment of 16-bit data irrelevant.  If the camera noise level is high enough to make this an acceptable procedure, then it has the additional advantage that it will preserve negative values in gain-normalized images.  Such values can occur and be meaningful in images with very low counts if the bias of the camera drifts downwards after taking a dark reference.

Save data as:

Allows data to be saved as bytes or 16-bit integers.  If there is a 16-bit camera and values are not being divided by 2, or if there are any buffers with unsigned data in them, an option to save in the non-standard unsigned integer mode is also available.

When saving 16-bit data:

Controls the treatment of unsigned 16-bit integer data being saved to a siged integer file.  The three options are to truncate at 32767, divide by 2, or subtract 32768, as described above.

Number of pixels to truncate converting to bytes:

Controls the number of pixels that will be truncated as black (0) or white (255) when images are scaled to bytes.

Images are captured as 16-bit integers, and the default is to save them to file in this mode.  Images can be saved as bytes (range 0 to 255), but this requires scaling of the intensity values into this limited range.  To keep the scaling from being dominated by a few extreme values (e.g., from X-rays), which would compress the dynamic range for the rest of the image data, the program will find a scaling which truncates a certain number of extremely dark and light pixels at 0 and 255, respectively.  Every image is scaled independently.  If you select byte storage, you can control the number of pixels that will be truncated with these text boxes.

Save in extended header:

Select whether to save tilt angle, C2 intensity value, stage position, or magnification in the header.

Maximum number of sections:

Indicate maximum number of sections that space is needed for.

The program can store various information for each picture in the ‘extended header’ of the image file.  The check boxes allow you to choose which information is stored; namely tilt angles, the stage position, and magnification.  If you are montaging, piece coordinates are automatically stored in the header as well.  Because these data are placed in the header, the program needs to know when first opening the file how much space to allocate for it.  This is the reason for the text box where you specify ‘Maximum number of sections’.  The admonition is to be generous because if you run out of header space your only choice is to start a new file.

Cancel:

Cancel file setup (and montaging, if setting that up).


No Help Available

No help is available for this area of the window.


No Help Available

No help is available for this message box.

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