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. Differences in operation in STEM mode are all described in a section at the end, Tilt Series in STEM Mode.
The dialog entries are organized into the following groups:
| Tilt Angle Specifications | Control the starting and ending angles and angular increment, unidirectional versus bidirectional series, the delay time after tilting, and angle-dependent parameter changes |
| Size and Image Shift Specifications | Control magnification, image shift resetting, beam centering, and zero-loss peak alignment. |
| Beam Intensity Control | Control beam intensity or exposure time changes. |
| Camera Selection and Options | Select the camera for the tilt series and set camera-related options |
| Autofocus Control | Control autofocus behavior. |
| Initial and Final Actions | Select options for startup phase of series. |
| Tracking Control Parameters | Set parameters to control prediction and tracking behavior. |
| Dialog Control and Action Buttons | Change appearance of dialog and select an action upon closing the dialog. |
Each of the groups (except the buttons on the bottom) can be opened and closed independently with the small +/- button in the upper left corner. In addition, the dialog control buttons allow you to step from one group to the next or to show the left side, the right side, or the full dialog (see Dialog Control and Action Buttons ). Each time that you open the dialog, it will be appear in a form similar to way you last left it: with the full dialog if the full dialog was showing before; with the left half if one half was showing before, otherwise with the first group open.
Tilt Angle Specifications
This grouping contains various controls related to tilting and tilt angle. The increment, delay time, and choice of cosine tilting duplicate the entries that can be made through the Tilt Control panel .
Start at / Tilt To
Enter the starting tilt angle in this text box for a unidirectional series, or the highest angle to go to on the first side for bidirectional or dose-symmetric series. This angle can be positive or negative. 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 or one based on previous tilt series. This value cannot be changed after a unidirectional tilt series is started, after a bidirectional series has finished its first part going to this angle, or after a dose-symmetric series has reached this angle and taken at least one image on the second side.
End at
Enter the ending tilt angle in this text box for a unidirectional series, or the highest angle to go to on the second side for other series. 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 for unidirectional or bidirectional series, but it cannot be changed after a dose-symmetric series has reached this angle and taken at least one image on the first side.
If there is a star after this text box when opening the dialog with the TS Params button in the Navigator window, it means that the angles you are seeing, which may apply to multiple tilt series, have been overridden by the 'SetItemSeriesAngles' script command for this Navigator item.
Swap
Use this button to exchange the starting and ending tilt angles in the two text boxes.
Base 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. Cosine tilting is not available for dose-symmetric series.
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.
Run series in two directions from
Turn this option on for either a bidirectional or a dose-symmetric series. A bidirectional series will start at the angle indicated in the text box, tilt up to the angle in the Start at text box (now labeled Tilt to), return to the starting angle, and tilt to the ending angle. If you want the series to start at an intermediate angle and go through zero degrees in the first segment, you must put an angle of the right sign in the text box (the same sign as the ending angle). The program will take a lower magnification 'anchor' image at the starting angle before it starts, and then realign to this image when it returns to the starting angle. Another special feature of this acquisition method is that if an image that is too dim is encountered at a high tilt in the first part of the tilt series, the first part will be ended and the second part begun automatically. By default, this happens if the angle is at least 55 degrees or within 5 degrees of the Tilt to angle, settings that can be adjusted in the Tilt Series Batch Policies dialog box.
If there is a star after this text box when opening the dialog with the TS Params button in the Navigator window, it means that the angle you are seeing, which may apply to multiple tilt series, has been overridden by the 'SetItemSeriesAngles' script command for this Navigator item.
Return with Walk Up
By default, the program will return to the starting angle of a bidirectional series by tilting back in steps and restoring the stage position and image shift at which the anchor was taken, with adjustments for backlash if possible. (Tilting in steps is probably needed only for side-entry holders and can be bypassed by setting the SerialEM property 'TSStepForBidirReturn' to 100. In that case, if a slower speed is desired for the return, the property 'TSSpeedForOneStepReturn' can be set with a speed factor.) However, if Return with Walk Up is checked, it will use the Walk Up task to return instead, but it will still realign to the anchor. This option should be used only if necessary. Particularly in Low Dose mode, there is some risk of failed correlations in the Walk Up procedure, as well as a risk that the position has already gotten off in the last few tilts. In fact, when the first part of the series is ended because of a dim image, the program assumes that the position is compromised and returns with steps even if Walk Up was selected. The main reason for using Walk Up would be if it is too difficult to get an anchor image with a large enough field of view.
Magnification for anchor
The magnification of the bidirectional anchor is selected with the spin button; the field of view at that magnification is shown to the right of the spin button. This magnification will be initialized to give at least an 8 micron field of view. This large a field of view may be necessary because the realignment to the anchor occurs after a large tilt and a long time interval after the anchor was acquired. The field of view will be shown in bold with two stars if it falls below 8 microns, and with 4 stars if it falls below 6 microns. (The property 'TSBidirAnchorMinField' can be used to set different criteria.)
Use View for anchor
When this option is checked, a View image will be used for the bidirectional anchor in Low Dose mode. If it is unchecked, then the program will allow you to select a magnification for Low Dose mode that is less than that of a View image, and it will use View parameters as the basis for taking that image. Do not assume that a View image will be reliable for the anchor just because it works for other operations.
Use dose-symmetric scheme
Turn on this option for a dose-symmetric series, where the program will alternate between the two directions of tilting based on parameters set in the Dose Symmetric Tilt Series Parameters dialog, starting at the angle specified in the Run series in two directions from text box. Background and details on this tilting method are available in Acquiring Tilt Series as well as in the help for that setup dialog. This is a form of bidirectional series, so this check box is not enabled unless the option for that is selected. Also, the dose-symmetric option is available only in Low Dose mode. Changes in this dialog that you will see after selecting this option are:
Set Up
Once a dose-symmetric series is selected, press this button to open the Dose Symmetric Tilt Series Parameters dialog and specify the sequence of alternations between the two sides of the series, as well as few other parameters.
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 and when autofocusing. As of SerialEM 4.0 (2/21/22), this delay is separate from the general tilt delay set in the Tilt Control panel, which is the one used when finding eucentricity at the beginning of a series.
Change some settings as a function of angle
Select this option if you want to make scheduled changes in some parameters at various points in the tilt series. The parameters that can be changed include Record exposure time and drift settling time, the target for autofocusing, and energy loss and slit width when using an energy filter. If you have already defined some changes, this option must be selected for them to be used during the series. For a dose-symmetric series, the tilt angles for making changes are all relative to the initial angle of the series.
Set Changes
Once you have enabled the use of scheduled changes with the adjacent check box, use this button to set, adjust, or view the changes in the Tilt Series Scheduled Changes dialog box .
Size, Image Shift, and Recentering 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.
At start and when needed, track at magnification X
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 to X microns
Enter the maximum image shift that should be allowed to accumulate before the program resets image shifts. For a dose-symmetric series, the limit shown applies only at the start of the series.
Recenter beam from Low Dose tracking images
This option is available in Low Dose mode for keeping the beam centered during a tilt series. When the program takes a new track reference or a tracking image before a Record, it will call the beam centering routine used with the Center Beam command . However, the beam will be moved only if the radius of the circle fit to detected beam edge is less than 90% of the diagonal distance from the center to a corner of the image, and if the error of the fit is less than 3% of this distance. These restrictions are an attempt to avoid mis-centering the beam when the edge of a grid bar is in the image. To use this option, you should set the intensity for the Trial area so the beam edges occupy all four corners; a size midway between those of the circumscribed and the inscribed circles should work. If necessary, the maximum radius allowed can be changed with the property TSTrialCenterMaxRadFrac, which is 0.45 by default.
Autocenter beam every X minutes
Select this option to have the beam autocentering routine that is used with the Autocenter Beam command run periodically during a tilt series. The option is available if autocentering has been set up for the magnification and camera being used for the series. Enter the interval in minutes between autocentering runs in the adjacent text box. The first interval is counted from the beginning of the tilt series because it is assumed that the beam was centered either manually or with autocentering just before the series.
Autocenter beam in second half of series at X degrees
Select this option to have the beam autocentering routine run once when the tilt series crosses back above the angle specified in the text box. This option allows the beam to be centered before it becomes constricted at high angles. Enter a positive angle even if angles are negative in the second half of the series. This option is unavailable for dose-symmetric series.
Refine the zero-loss peak every X minutes
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 the specimen exposure 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. Traditionally, SerialEM has used changes in beam intensity (brightness, C2 or illuminated area on Thermo/FEI scopes, or C3 on JEOL) to compensate for changes in beam attenuation. It is also possible to have exposure time changed instead. Changing exposure time only will leave the beam in a constant state, which would allow one to stay in parallel illumination. However, increasing exposure time may lead to drift in the images, and for plastic sections, it will generally require longer drift settling times. If you have a direct electron detector, changing exposure time presents additional complications because it may be constrained to be a multiple of a certain value.
When doing a dose-symmetric series, the changes described in this section that depend on angle (cosine variation and tapering of counts) are done relative to the starting angle of the tlit series.
Change Record exposure time instead of beam intensity
Select this option to vary the exposure time of Record images instead of beam intensity to achieve whatever control of specimen exposure is specified in the options below this. When using this alternative with a CCD camera where dark references are specific to the exposure time, the program will use interpolation between dark references, if possible, to avoid having to take a new dark reference for each image. This is the same interpolation that can be activated with the Interpolate Dark Refs command in the Camera menu, and the program will try to use this feature regardless of whether you have selected it in that menu. However, interpolation cannot be used if you are not gain-normalizing images in SerialEM, and in that case a dark reference will be taken for each Record image if this option is selected.
This option is not available if you have scheduled changes in exposure time, which means that both exposure time and intensity might change during the series. Specifically, you might have a set of discrete exposure changes that occur at intervals during the series. Between those changes, the beam intensity will be changed to achieve whatever control of specimen exposure you have specified. When the exposure time changes, the beam intensity will be changed to compensate and maintain the desired specimen exposure.
If you have a K2 camera, this option is problematic because the exposure time is constrained to a multiple of 0.1 sec or 0.5 sec in counting or super-resolution mode without Dose Fractionation, or to a multiple of the frame time with Dose Fractionation on. With such constraints, the program will likely not be able to change exposure time at fine enough increments. It issues a warning whenever it cannot set the exposure time within 5% of the intended level. There are two realistic alternatives here. One is to set up scheduled changes in exposure time with the Tilt Series Scheduled Changes Dialog, which will pick exposure times that meet the constraint and angles at which those times should be set. The program would then change intensity between exposure time changes. The second alternative is a hidden workaround to the constraints: set the frame time equal to the exposure time and turn on Dose Fractionation mode without aligning or saving frames. The program will then change exposure time and frame time together in multiples of 2.5 ms. However, acquisition will take longer, 4 sec in counting mode or 5.5 sec in super-resolution. This alternative works only if you have not explicitly scheduled any changes in frame time.
All camera parameters that are varied during a tilt series are restored to their original values when the series is terminated.
Keep beam intensity constant
Select this option if you do not want the program to change beam intensity or exposure time during the series. Unless you are in low dose mode, the program will actually do nothing to keep the intensity or exposure time constant, and you are free to adjust it yourself.
Vary intensity to be x higher at 60 deg (1/cos to 1/y power)
This option allows you to vary the beam intensity or exposure time so as to achieve a selected increase from zero to 60 degrees. The increase will be computed as 1 over the cosine of the tilt angle, raised to an inverse power. Prior to serialEM 3.9, this increase was specified as powers from 1/1 to 1/5; now the setting is more flexible and more intuitive. With an increase of 2, corresponding to a power of 1, this option would vary beam intensity or exposure time 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. Both lower and higher increases than this can be selected.
Set intensity to keep mean counts of Record images at
Select this option to have the beam or exposure time 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 to X above +/- Y degrees
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 (or, alternatively, if the exposure time cannot be increased above a certain point without getting too much drift in the image). 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 intensity below current value (use if intensity already set up)
If you are starting at high tilt and have already set up the intensity or exposure time that you want at the starting tilt, use this option instead of specifying tapering of counts. This option will maintain the intensity or exposure time 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 or the exposure to be increased 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 or exposure time is the value set just after saving the first image of the tilt series and adjusting the beam or exposure time 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 or exposure was set up to be appropriate at zero tilt. When the tilt series starts, intensity or exposure will be increased by the appropriate inverse cosine factor.
Dose Estimate
When constant or 1/cosine intensities are selected, information about the total dose will appear, based on the tilt angle range and increment and some of the other settings in this group, and on the current Record parameters and beam settings. If electron dose is calibrated, the line will show the expected dose at zero and high tilt and the total dose in electrons per square Angstrom. If dose is not calibrated, the line will show the dose as a multiple of the dose at zero and at high tilt.
Change other exposures proportional to Record change
This option is available when you have either scheduled changes in Record exposure times or chosen to change exposure time rather than beam intensity. Select it to have the exposure times for all the other camera parameter sets changed whenever the Record exposure is changed, by an amount proportional to the change in Record exposure. Note that if you stop the series, you are free to change these exposure times in the Camera Parameters dialog box, although you may be prevented from changing the record exposure time.
Change drift settling proportional to exposure change
Select this option to have drift settling changed proportional to a change in exposure time whenever an exposure time is changed for a camera parameter set. The option is available if all exposures are being changed via the option just above, or if just Record exposure is being changed and there are no scheduled changes in Record drift settling. If there are such scheduled changes, they will control the Record drift settling regardless of whether this option is selected. If you stop the series, you are still free to change the settling times in the Camera Parameters dialog box.
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.
Average Record dark references
This 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 in the Camera Parameters dialog.
If taking frames, get initial partial sum of N frames
This option is enabled for a K2/K3 camera and allows an "early return" when either saving frames or aligning frames in the SEMCCD plugin to DM. An image with the sum of the selected number of frames will be returned as soon as those frames are available, and the program is free to go on to other tasks. The initial sum is used for alignment and is also saved to the output file as a placekeeper. The full sum is retrieved just before the next Record image and saved to the file. None of this will occur if there is extra Record output, when montaging, or if any other relevant camera parameters sets have dose-fractionation turned on.
This option may produce modest time savings if the frame operations are relatively time-consuming, such as by using super-resolution mode, taking many frames, or saving with compression, and if the program needs to do some operations between Record images. There may be no speedup with counting mode images.
Autofocus Control
The first four parameters are synchronized with the corresponding values set in the Focus/Tune menu: they are initialized with the values set there, and setting a new value here will change the value used in the program generally, except when setting parameters for a batch acquisition.
Defocus target
The target defocus value for autofocusing will appear in this text box and a new value can be entered. If the label after this text box is 'um*' when opening the dialog with the TS Params button in the Navigator window, it means that the angle you are seeing, which may apply to multiple tilt series, has been overridden by the 'SetItemSeriesAngles' script command for this Navigator item.
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.
Iteration threshold
The threshold change for the autofocus routine to do another iteration will appear in this text box and a new value can be entered.
Do autofocus at least every X degrees
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.
Skip autofocusing
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.
Focus every time above X degrees
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. With dose-symmetric tilting, the starting angle of the series will be added to the specified number to determine the actual threshold angles.
Repeat autofocus if focus differs from prediction by > X um
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 when error in focus prediction exceeds X um
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.
Check autofocus, stop if measured < N X actual defocus
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.
Stop if autofocus exceeds absolute focus limits
This is one of two options for limiting the focus changes that occur during a tilt series. This option causes autofocus to fail, original focus to be restored, and the tilt series to stop if focus exceeds the absolute limits around eucentric focus that are set with the Set Absolute Limits command in the Focus menu. It is most suitable for preventing focus from being set in a range that would activate the Falcon Dose Protector. It applies to any autofocus done at the start or during the course of the series.
Stop if autofocus change exceeds X um
This option can be used to protect against large changes in focus, such as when a grid bar or piece of contamination comes into the focusing area. If the focus change imposed by autofocusing would exceed the amount entered in the text box, the autofocus will fail, focus will be restored to its original value, and the tilt series will stop. This option also applies to any autofocus done at the start or during the course of the series.
Initial and Final 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.
Align to image now in Buffer A
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. The program will rmaintain alignment to the specimen position with the Refine & Realign procedure.
Leave mid-tilt anchor near plus or minus X degrees
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 that you have left in buffer X
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.
Close column valves at end of series
This option provides the final control of beam intensity by allowing you have the column or gun 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. On a JEOL without a FEG, this option can be used to turn the filament off instead.
Tracking Control Parameters
This grouping contains various parameters that govern the Tilt Series Controller's behavior with regard to predictions for positional changes and the tracking steps that it will do.
Wait for drift to settle
Select this option to run the Wait for Drift task, and press the Set Up button to open the Wait for Drift Setup dialog and set parameters that control both the operation of the task and at what angles it is run during tilt series. This task is run between initial tracking and autofocusing. Unless you have selected to track only after focusing, any drift measured by the routine will be corrected with image shift to maintain specimen alignment during the procedure. (The setting to correct drift in the Wait for Drift Setup dialog is ignored here.) The task is integrated into the tilt series operations to the extent possible, depending on the type of image that you choose for measuring drift:
1) If drift is measured with Trial images, and an initial tracking image was taken, that image is used as the first one for measuring drift. This happens regardless of whether you have set a different binning or exposure for the drift measurement. In addition, when you have not set different parameters for the drift Trials and have selected tracking before focusing, a tracking image will be taken even when the predictions indicate no tracking is needed; it will be aligned with the reference then used as the first image of the drift measurement.
2) If drift is measured between the the first and third image of autofocus runs, then the results from the last run will be used to evaluate whether focusing is still needed. On the one hand, if that run satisfies the criteria that would be used for a run by the Tilt Series Controller, then autofocus will be skipped. On the other hand, if the focus change in that run exceeded the threshold for iterating within the autofocus routine, autofocus will be done even if predictions indicated that it could be skipped. (Iterations with the autofocus routine are suppressed when it run to measure drift).
Repeat Record if percentage of field lost is more than
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. In low dose mode, the next option determines what happens if this option is selected.
Stop and ask before repeating Record in Low Dose Mode
With this option and the preceding one selected, the program will stop and ask for confirmation before repeating a Record image in low dose mode. This was the behavior before this checkbox was added. If this option is turned off, the program will retake the image up to three times even in low dose mode. This is useful if you are using low dose mode to get different magnifications for tracking or focusing and the extra exposure of the specimen is not an issue.
Stop if Autoalign shifts more than X % of image size
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 error in X/Y prediction is > X % of field
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 to allow manual adjustment
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, not Record 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 by > X %
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.
Dialog Control and Action Buttons
Previous - Next
Press these buttons to close up the last group of controls currently open and open up either the previous or the next group, respectively.
First Half - Second Half
Press these buttons to see the left or the right half of the full dialog, respectively. If you close the dialog in either of these states, it will reopen showing the left half.
Full
Press this button to see the full dialog as it appeared in earlier (2.x) versions of SerialEM. If you close the dialog in this state, it will reopen as a full dialog.
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. When the dialog is opened to set parameters for a series to be run from the Navigator, this button is converted to Set Up Extra Output. Use this button to open the Tilt Series Extra Output dialog and set or change the selections for extra output.
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. This button is also not present when the dialog is opened from the Navigator.
Postpone/OK
When the dialog is opened from the menu, this button ('Postpone') 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. When the dialog is opened from the Navigator, this is an 'OK' button for closing the dialog and saving the parameters.
Cancel
This button will close the dialog box and discard any parameter changes that you have made.
Tilt Series in STEM ModeWhen the dialog is entered with the microscope in STEM mode, or if the STEM camera is selected, several features changes, reflecting intrinsic differences in tilt series acquisition from TEM mode.
Shift/Centering Options and Interset Shifts. Options to control beam centering and energy filter alignment are disabled. In this section, a line is added to report whether image shifts exist to keep Trial and Focus images centered on the Record images. These shifts can be calibrated quickly before a tilt series, once the exposure times have been finalized. See Assess Interset Shifts for details.
Exposure Time Changes Only. It is not possible to change beam intensity continuously in STEM mode, so the Beam Intensity Control section allows changes only in exposure time. Moreover, image counts do not scale with exposure, so options for maintaining a constant number of counts are disabled. This just leaves the option for changing exposure as a function of the cosine of the tilt angle. There are two situations where this would not be appropriate. First, dark field signals may become stronger with increased sample thickness rather than weaker. Second, if you are using dynamic focusing with Thermo/FEI STEM, you may need to use a discrete set of Record exposure times where the flyback time has been calibrated (see Quick Flyback Time), rather than allowing exposure time to vary continuously. The solution is to check 'Change some settings as a function of angle', press Set Changes, and enter the needed exposures in the Tilt Series Scheduled Changes dialog box.
Autofocus Differences. The 'Skip Autofocus' checkbox is enabled in STEM mode, as in Low Dose mode, in case autofocus is not needed or does not work (e.g., in microprobe mode on a Thermo/FEI). The option to check autofocus is disabled.
Separate Parameters. For several parameters, separate values are maintained for TEM and nanoprobe STEM mode, and microprobe STEM mode (if present), and stored in your settings file. These parameters are the tilt series magnification, the magnification for low mag tracking, and the maximum focus prediction error allowed before autofocusing is needed. The latter is maintained separately because STEM imaging in nanoprobe mode probably requires a tighter tolerance on focus than TEM imaging.