Calibration menu commands

The Calibration menu and its submenus offer the following commands:

Image Shift command (Calibration - Image & Stage Shift sub-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 1024 pixels for direct detector cameras or 512 pixels for other cameras, using an exposure time based on the settings in the Trial parameter set.  This binning can prevented by turning on the Use Trial Binning option to make it use whatever binning is in the Trial parameter set.  Similarly, full-field images will be taken unless Use Trial Size is turned on.  It is important that the images have sufficient detail and enough counts to give reliable correlations.  Either kind of  deficiency will be reflected by a low cross-correlation coefficient (CCC, reported after the procedure); it is best to keep the CCC above 0.5.

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. On Thermo/FEI scopes, there are two magnification ranges for image shift calibrations: LM mode mags and all higher mags.  On JEOL and Hitachi scopes, there will typically be additional boundaries defined between smaller mag ranges.

At the end, you will see messages about the minimum and mean CCCs, 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.  On the other hand, if the calibration is bad enough, you will be asked if you want the program to forget about it, which is usually a good idea.

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 .  Also, if there is a lot of distortion, the procedure may report a relatively poor result; this is common in the bottom two SA magnifications of a Krios in EFTEM mode.  A remedy here is to use a half area, or even a quarter area if necessary.  Turn on Use Trial Size, adjust the binning if necessary so that there are still ~500 pixels, and increase the beam intensity to compensate for the loss in image area.

IS from Scratch command (Calibration - Image & Stage Shift sub-menu)

This command will calibrate image shift in two rounds (13 pictures), without relying on any existing image shift 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. It does rely on the program's current notion of the pixel size at the current magnification, and can fail if that notion is way off.  The pixel size will be based on either a calibrated pixel size for this magnification entered on a 'RotationAndPixel' line for this camera, or on the FilmToCameraMagnification value for this camera.  If you have trouble with this procedure, start at a magnification where you can run the Find Pixel Size command successfully, and do so before starting this calibration.  The program will give you a chance to use the pixel size from that routine if it differs enough from the one indicated by the FilmToCameraMagnification value.

Stage Shift command (Calibration - Image & Stage Shift sub-menu)

This command will calibrate the relation between stage movements and position on the camera at a particular magnification. This calibration requires moving the stage over relatively large distances, not just because small movements would be inherently inaccurate, but also because the stage may have systematic and periodic inaccuracies. In the Compustage, the distance actually moved per 1 micron of requested movement can be less or more than 1 micron by as much as ~15%, and the pattern of the error repeats every 40-60 microns (an effect first described by Pulokas et al., 1999). Stage calibration is done over a defined distance, taking up to 15 steps if needed to cover that distance. The distances in X and Y are set with two properties, StageCycleLengthX and StageCycleLengthY, whose defaults for Thermo/FEI scopes (62 and 41 microns) are based on the values found on the two Tecnais in Boulder, which match those found in Pulokas at el., 1999. See Other Microscope Calibrations for instructions on assessing the cycle length. Even if you do not have the right cycle length, the calibration should be fairly accurate because it is done over a long distance.  This effect is apparently minimal on JEOLs and the Hitachi HT7700, so the default there is 40 microns.

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 it at the lowest useful magnification not in LM mode. It can be done at a higher magnification instead, but that will require more steps. If the Navigator is going to be used, the calibration should be done at 2 or 3 mags in LM mode, such as the highest one, an intermediate one, and one near the lowest mag where whole grid montages would be done. If the image on the screen appears unstable in LM mode (presumably due to charging effects), try using the smallest objective aperture that does not occlude the image on the camera.

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. The rotation angle between the lowest M-mode mag and the highest mag in LM mode should also be entered, unless whole image correlations are being used.

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. On an older Thermo/FEI scope, if the calibration is done over an area that fits within one grid square of the replica grating, there is a good chance that it will be off by 10%.  On a JEOL, where the cycle length is probably not relevant and a correlation over only 20-25 microns may be accurate enough, the replica grating can be used, unless calibrating with a camera on an energy filter (see the Whole Image Corr command).  Also, the replica grating could be used on a Thermo/FEI scope at a mag low enough so that there are several grid squares in the field, provided that there is enough visible difference between the grid squares. Aside from this, correlations may not work when the image contains grid bars. There need to be image features useful for correlation throughout the region that will be used (~60 microns). Position the specimen in the middle of such a region before starting the calibration. If you have difficulty getting a specimen area large enough for the calibration, you can limit its extent with the property MaxStageCalExtent.

After you start the procedure, the program will tell you how many pictures will be taken and the total distance over which the calibration will be done. It will take a series of pictures with movements in X, then a series with stage movements in Y. After each picture except the first in each series, it will toggle for a few seconds between two images that are supposed to line up. Watch and make sure that the images do line up. If not, stop the calibration. At the end it will report the transformation matrix, the maximum range for the multiple estimates of each factor, and the implied transformation from stage to specimen coordinates (provided that there is sufficient information to derive the latter). The variation between estimates should be less than 10%, and the stage to specimen transformation should be close to -1.0 0. 0. -1.0; if not, you should redo the calibration unless you are sure that the paired images all lined up well. The stage to specimen transformation can differ from -1 0 0 -1 if there is an error in the rotation angle for this magnification derived from the entries in the properties file (e.g., a 10° error will result in middle terms of ±0.17).

The calibration also reports an estimate for the rotation angle of this magnification; this appears to be accurate within 0.2-0.5 degrees.

Whole Image Corr command (Calibration - Image & Stage Shift sub-menu)

This commands toggles whether the image correlations used for stage calibration are done over the whole image area or just over the region of expected overlap.  Whole image correlations are on by default because they work robustly with non-periodic specimens and make the calibration procedure immune to large errors in the image rotation angles specified in the properties file.  When correlating just the region of expected overlap, the procedure will fail if these angles are off by more than 40 degrees. Whole image correlations also work robustly with a cross-line grating with latex beads, provided that there are enough beads in the field and that image distortions are not too large.  They will not work when calibrating GIF images with the grating, the overlap-only correlations are more reliable but can also fail in that case.  Use a non-periodic specimen for stage calibrations on a GIF.  The whole image correlations can also fail at very low magnification if using the cross-line grating, because the grid bars can dominate the alignment.  Use a specimen with larger grid squares and keep this option on, or turn this option off and make sure that the image rotation angles are approximately correct before calibrating the lowest magnifications.

Set Cycle Lengths command (Calibration - Image & Stage Shift sub-menu)

Use this command to set the cycle lengths for stage calibration, which determine the distances in X and Y over which the calibration is run.  Specifically, the calibration is run over this distance, if possible, at higher magnifications, and over a multiple of this distance at lower magnifications when only three images are being acquired in each direction.  This setting is temporary, not saved when the program exits; change the properties 'StageCycleLengthX' and StageCycleLengthY' to make a permanent change.  The cycle lengths should not be changed for Thermo/FEI scopes except for experimenting, unless it is found that the systematic variation in stage movement is minimal with a particular stage.

Use Trial Binning command (Calibration - Image & Stage Shift sub-menu)

If this item is checked, then image and stage shift calibrations will take images at the current binning of the Trial parameters instead of adjusting the binning and exposure time to achieve a target size.  This item is initialized from the property 'UseTrialBinningForShiftCal' and is not saved in user settings between sessions.  Using a lower binning can be useful for achieving a good signal-to-noise ratio without saturating the camera.  It will not make the cross-correlations more prone to failure, because images are binned again before correlation if necessary.

Use Trial Size command (Calibration - Image & Stage Shift sub-menu)

If this item is checked, then image and stage shift calibrations will take images at the current size of the Trial parameters instead of taking full-field images.  This item is initialized from the property 'UseTrialSizeForShiftCal' and is not saved in user settings between sessions.  Although the calibrations generally have no trouble with non-square cameras, this setting might be needed for side-mount cameras where there is too much distortion outside of a square region, or for a camera with a bad area on one side.  To accommodate the latter situation, subareas will also have the same offset from center, if any, as the Trial parameters.

List IS Vectors command (Calibration - Image & Stage Shift sub-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, unless using projector shifts (PLA) for image shift on a JEOL, in which case they should decline continuously within a magnification range.. 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 - Image & Stage Shift sub-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.  Often when this is not the case, the first and last terms have a similar absolute value and the second and third terms have a similar absolute value, all corresponding to the sine and cosine of some angle (e.g., -0.85  0.52  -0.47  0.87). This pattern indicates that the calibration is probably good but that the program has an incorrect notion of the absolute rotation angle at the given magnification.  Transformations that do not fit this pattern are generally bad.

Remove IS Cal command (Calibration - Image & Stage Shift sub-menu)

Use this command to remove one image shift calibration for the current camera.  The program will ask for the magnification of the calibration, then show you the matrix and ask you to confirm the removal.  Use List IS Vectors first to see which calibrations have a vector length or axis angle clearly different from the rest.  When an image shift calibration is done, it is saved automatically in the short term calibration file (SEMshortTermCal.txt) for a day, so even if the calibration has not been intentionally saved to the main file (SerialEMcalibrations.txt), it can linger to have bad effects.  This command can be used to remove its effects instead of rerunning the calibration from scratch.  The consequences of the command depend on whether the particular calibration has been saved to the main file:

• Bad calibration saved to main file: You must save the calibrations again to remove it fully; otherwise it will reappear when you restart the program.  You must redo the calibration at some point to have a calibration for that magnification.
• Bad calibration not saved to file: You do not need to save calibrations, but if you do, they will not include this calibration.  Restart the program to revert to a previous calibration, if any.

Remove Stage Cal command (Calibration - Image & Stage Shift sub-menu)

Use this command to remove one stage shift calibration for the current camera.  This command works just like Remove IS Cal, except that here you would use List Stage Cals first to see which calibrations have a specimen to stage matrix clearly different from the rest. 

Mag IS Offsets command (Calibration - Image & Stage Shift sub-menu)

Use this command to determine image shift offsets that will keep a feature centered as magnification is changed. This calibration may be needed to get the best performance from the Navigator, such as when a position is marked on an image at one magnification and then sought at a much higher magnification. However, the image shift offsets for View and Search in Low Dose mode, and the mechanism of taking anchor maps at the same position as a target to be aligned to, provide good alternatives to having this calibration.  The disadvantage of the calibration is that it gets off over time more easily than other calibrations, and becomes invalid when magnification alignment in the scope itself is redone.  It should be done only if someone is committed to keeping it up to date, otherwise the Navigator will apply incorrect shifts that may be worse than the uncorrected intrinsic shifts. 

One of the limitations in determining reliable offsets is the degree to which the image shifts when the beam is moved, which can be particularly severe in LM mode depending on the focus setting. To minimize the effect of this shifting, you will be advised to keep the beam centered in LM mode during this calibration. More importantly, the program will go to a standard focus in LM mode if one is defined. On a Thermo/FEI scope, this is defined by default to be at 'eucentric focus'; on a JEOL, a numeric value for the standard focus needs to be determined with the 'ReportFocus' script command and entered as the property StandardLowMagFocus.

It is recommended that the offsets be calibrated from the highest magnification where there is an obvious misalignment between magnifications, down to the lowest magnification to be used for Navigator mapping. The entire range does not need to be done at once. The calibration can be done just over parts of the range where the misalignments are bad. If the offsets are recalibrated over a subset of the range, the new values will be merged in with the old ones determined for the other magnifications.

To do the calibration, you need a specimen with a feature that can be centered over the full range of magnifications to be calibrated. The corner of a section works well for this. Before you start, set up the beam to a reasonable size and brightness and turn on Intensity Zoom if available. Do this in both regular mag and LM mode. Set up Trial parameters to give an adequate image for centering the feature. Go to the highest magnification to be calibrated and start the procedure. You will see a message box with instructions, then you will be asked whether you want to take a trial image automatically at each new magnification. This is convenient if Intensity Zoom or an equivalent intensity adjustment is on, but may be problematic otherwise.  Continuous acquisition mode can also be used for the routine, and there will be blanking during the magnification changes.  If you use continuous mode, the program will skip the Trial images.  Use the space bar to stop continuous mode without stopping the routine.

This calibration routine is nearly unique in that it allows you to operate the program and waits for notification that an image is aligned. It converts the first Script button in the Camera & Script Control Panel into a 'NextMag' button that you press after an image is aligned. The 'End' and 'STOP' buttons in that panel are used to stop the procedure. The distinction between them is that 'End' will record an image shift for the current magnification whereas 'STOP' will not. 

After you start the routine, center the image feature by shifting with the right mouse button. You can use stage shift at this point if necessary, but thereafter you must shift only with image shift. (The option for moving the stage for big mouse movements is disabled during the procedure to prevent stage shifts.) At each magnification, center the feature then press 'NextMag'. The program will make sure that you are at the correct magnification, record the image shift, lower the magnification, and take a picture if you selected that option.

When you step down into LM mode, try to keep the objective aperture in, switching to the largest aperture when necessary, then taking out the aperture when necessary. On both an F20 and an F30, this has been seen to eliminate strange charging effects that can affect image size and position.

One magnification must be set as the reference where the image shift offset is zero. After you end the routine, you will be asked which magnification should have its offset set to zero. You should specify the magnification typically used for tomography, so that there will not be large amounts of 'hidden' image shift there. If you calibrated only below that magnification, you can specify the top of the range that was calibrated and achieve the same result.

If you have both a pre-energy filter and an energy filter camera, the setting of a reference is more complicated. Once an offset between the two cameras has been defined, only one camera can have a magnification with an offset of zero, so you will be asked to specify which camera this is to be.

This calibration requires that image shift be calibrated at least once in every defined magnification range. On a Thermo/FEI scope, this just means you need at least one image shift calibration in LM mode, in addition to the many that you should have done in regular mag mode. On a JEOL, this may mean that you need to calibrate image shift at every mag of interest, then use 'List IS Vectors' to determine image shift boundaries, and define those boundaries with the 'OtherShiftBoundaries' property.

Once there is an image shift offset calibration, you can turn on 'Adjust image shift between mags' in the Image Alignment & Focus control panel. The calibration routine will work with this option on, and recalibration might be easier with it on.

EFTEM IS Offset command (Calibration - Image & Stage Shift sub-menu)

Use this command to calibrate the image shift offset needed to align images from a pre-energy filter and energy filter (EF) camera. If you have such cameras, the recommended initial procedure would be to calibrate the image shift offsets on the regular camera, then run this command, then calibrate the offsets on the EF camera. However, they are independent procedures and you can run them in any order, and rerun one without having to rerun the others.

This command gathers information from images that you have already collected, so when you start it, it will tell you what you need to have done already and ask if you done it. Here is the procedure:

1. Go to a magnification not in LM mode on the pre-EF camera and center a feature. If you use mouse shifting to center the feature, take a final image that has no mouse shifting.
2. Copy this image to the first buffer after the buffers that roll, the usual autoalign buffer. (When you start the routine it will tell you what buffer it expects this image to be in.)
3. Go to EFTEM mode and take an image. Center the feature using autoalignment or mouse shift only, not stage shift.
4. Invoke this command. If you have not already specified which camera should have one magnification with image shift offset set to zero, you will be asked about this.

Set IS Delay Factor command (Calibration menu)

Use this command to set a factor for scaling the delay that occurs after an image shift.  This change applies only during this session of the program; a permanent value can be set with the property ISdelayScalingFactor.  This delay is imposed because it takes some time for image shift to settle at the target position, particularly on older Thermo/FEI scopes.  Image shift delay was re-evaluated in February 2022, leading to some new recommended defaults and the ability of the program to adopt the new defaults automatically in some cases.  These changes are described in ImageShiftDelays, which should be read thoroughly before trying to adjust delays.  You should also make sure that there is a roughly accurate value for the camera property 'StartupDelay' for any cameras where image shift settling is an issue.

The new way to assess delays involves using a K2 or K3 camera and taking frames while image shift is applied with the FrameSeriesFromVar command.  (Minor program changes would be needed to use other frame-saving cameras for this.)  A typical script is:
   var = { 0.1 0 0 5 2 0 }
   FrameSeriesFromVar var 20 0.1
   Record

where Record is set up to save frames with a 5 sec exposure and 0.08 sec frame time, and pixel size is around 1 Angstrom.  The '2' that is the next to last number in the 'var' line represents the desired image shift in X to test; a shift in Y could be used instead.  If the specimen is not already stable, one or more conditioning shots should be done with no frame saving.  After the script is run:

•  The frame file is opened in 3dmod to find the first frame at the new position, e.g. 18.  If the shift is not in the first third of the frames, you need to adjust the StartupDelay in the camera properties; if the shift is too early, adjust oneof  the '0.1' delays in the script.

• Alignframes is run on unnormalized frames with a command such as:
     alignframes -out junk.ali -vary 0.05,0.06,0.7,0.08 -pai 13 -titl -fra 18,63 -xf 2.xf 001.tif
  where the '-titl' is added to read gain reference and defect files from the file header and '18' was observerd to be the first frame after the shift. This outputs a file of shifts in '001.2.xf'.

• The curve is examined with
     tomodataplots -type 3 001.2.xf
  and the point is found where the drift has settled to roughly 1 Angstrom/sec.

The old way of assessing the need for more or less delay was to run the Image Shift command in the Calibrate menu.  First set the debug output key letters to anything other than 0 (1 will do).  Also, turn off Post-actions in the Camera menu. Calibrate image shift and at the end it will print "Drift converted to IS in nm:" followed by 4 lines computed from each of the 4 times that the calibration returns to zero image shift.  If image shift is perfectly settled and drift is constant with components Xdrift and Ydrift in the X and Y directions, then the 4 lines would all be similar and show Xdrift and Ydrift.  If image shift still needs to settle by an amount Xsettling or Ysettling, for shifts in X or Y, then the lines would have the form:
   Xdrift + Xsettle  Ydrift
   Xdrift - Xsettle   Ydrift
   Xdrift                Ydrift + Ysettle
   Xdrift                Ydrift - Ysettle

The amount of shift due to inadequate settling is reflected by the differences between the first two X numbers, and between the last two Y numbers. For example, with good settling and some drift, the numbers in one set of tests were:
-1.6 1.4
-1.8 1.2
-1.5 1.3
-1.5 1.0

Here, the differences reflecting settling are minimal (0.2 and 0.3) and are probably just due to the fact that drift was declining during the run.  With a reduced delay factor, these numbers became:
-0.2 0.5
-1.2 0.3
-0.2 0.9
-0.5 -0.5

Here the X difference is 1.0 and the Y difference is 1.4 - image shift has not settled completely.  With virtually no delay factor the differences become extreme (8.3 and 10.7)and no longer fit the simple model above because effects linger from one pair of pictures to the next:
 2.6 0.1
-5.7 0.1
 2.5 3.7
-0.1 -7.0

The protocol is thus to change the delay factor and run the calibration repeatedly, looking for the delay factor at which the X and Y differences due to settling either become very small or stabilize.  Do this at several magnifications (e.g., 5000, 15000, 25000) to make sure the scale factor is appropriate over a range of mags.  On a JEOL, there may be differences that appear to be due to incomplete settling, but if the differences remain constant as the scaling factor is increased, then the lowest factor at which the differences have these stable values is appropriate.

Note that this method cannot detect settling times less than the startup time of the camera, the time from when an image is requested to when the exposure starts.  The 0.8 sec adjustment mentioned in ImageShiftDelays as the default for the property 'StartupForISDelays' derives from the fact that the table was first prepared using a camera with that startup time.

Find Pixel Size command (Calibration - Pixel Size sub-menu)

Use this command to analyze an image of the cross-line replica grating in buffer A and determine the pixel size.  If buffer A contains a montage center and B has a montage overview, B will be copied to A and analyzed.  The program will take a filtered autocorrelation, which produces a square grid of correlation peaks with the highest peak at the center, where the image correlates perfectly with itself. It finds a starting peak close to the center, then locates peaks progressively farther out so that it can estimate the spacing between peaks from as many intervals as possible. Next it searches for a peak 90 degrees away from the starting peak, and finds peaks progressively farther out along that line. It averages the interval between peaks in the two directions and then computes a pixel size.

When it is successful, this routine shows the autocorrelation in buffer A, and leaves the green marker on the starting peak. It writes one line to the log window showing the mean spacing and number of intervals in each direction, and the size of the pixels in the image that was analyzed, which will be a binned pixel size if the image was binned. It writes a second line with the unbinned pixel size, although if the image was read from a file it will label this as 'APPARENTLY unbinned'. If the image was not read from a file, a final line will be written with the magnification index, unbinned pixel size, and magnification.

Images larger than a certain size are binned down to improve the signal-to-noise ratio for the analysis.  By default, the target size is picked automatically when using a cross-line replica grating; see the Set Binned Size command for details. The program will output a line indicating the target size and the number of blocks that it expects to appear in the image.  When first getting started, watch carefully that the number of blocks is close to correct; if it is off by much, it means that the program's current assumptions about pixel size are substantially off.  The best thing to do in that case is to make sure the correct peak has been found, exit the program, and insert the pixel size for this magnification into a RotationAndPixel line in the camera properties.  This better pixel size should make the routine pick the right binning for the whole mag range.

Reported distances are in terms of pixels in the original image, but if you look at the actual number of pixels between peaks it will be smaller by the binning factor.  Also note that dark regions are trimmed out before analysis.

Always make sure that the correct starting point is picked - it needs to be one of the points closest to the center peak, not one on a diagonal, or one between the strong peaks. Zoom up if needed so that you can tell which point is the central peak. If the point is incorrect, click a correct starting point and use the Try Again with Marker command described next.

Try Again with Marker command (Calibration - Pixel Size sub-menu)

Use this command when the Find Pixel Size command fails to show the correct starting point in the autocorrelation. With the autocorrelation still in buffer A and the grating image in buffer B, click with the left mouse button on one of the four points that are nearest to the central peak of the autocorrelation. Then select this command. The program will use the peak nearest to the marked point as the starting peak, but will still have to find a peak 90 degrees away from it for measuring the other direction.

Set Binned Size command (Calibration - Pixel Size sub-menu)

Use this command to set the maximum size that images will be binned to when running Find Pixel Size. The default value is 0, which means that the program will automatically select a binned size based on the expected number of blocks in the image of the cross-line replica grating.  (Sizes of 1024 or 512 are used when doing catalase or grid bars, respectively.)  Higher binning helps when there are relatively few peaks at higher maginifications, and also when the peaks appear weak relative to the central peak.  If you have to set a specific size, try 512 first and adjust as needed.

Add n*90 to a Rotation command (Calibration - Pixel Size sub-menu)

Use this command to adjust one of the rotations reported by List Relative Rotations by a multiple of +/- 90 degrees.  Enter the camera index, the magnification index, and the amount to add to the reported angle.  It may be more convenient to use this method, at the time when the magnification in question is being examined, than to adjust angles when getting a final listing.

List Relative Rotations command (Calibration - Pixel Size sub-menu)

Use this command to get a list of RotationAndPixel lines based on information determined with the pixel size finding routine. This information is saved between sessions of the program for up to 24 hours in the file SEMshortTermCal.txt.  A line will be output for each magnification where the pixel size was also measured at the next lower magnification; i.e., where a relative rotation is available to be included in the line. However, the relative rotation values will sometimes have to be adjusted by 90 or even 180 degrees, either when there is a large rotation or when the difference is reported to be large for a small rotation. Whenever the difference is large, the program will also print an alternative difference nearer to zero, which requires the line to be edited manually. The program will also print the product of measured pixel size and nominal magnification (p): this value is expected to be similar for each magnification on the same camera. If there are outliers, the pixel size calibration needs to be checked.

Catalase Crystal command (Calibration - Pixel Size sub-menu)

This command toggles whether the pixel size routine assumes that the image is from a catalase crystal instead of from the replica grating.  In this mode, it will look for the closest-spaced regular peaks, which should correspond to the 6.85 nm crystal spacing.  After picking that closest peak, it will look for the peaks 90 degrees away from that direction, which will be at twice the 8.75 nm crystal spacing.  Thus, it will report finding many fewer peaks along that line than along the short axis.  The routine has not been optimized for this specimen.  Make sure that it finds the closest peak in the correct direction, and mark that one if necessary.  If it fails, try reducing the binned size to 512 with the Set Binned Size command.

Mesh for Grid Bars command (Calibration - Pixel Size sub-menu)

When at least two grid bars appear in the image in each dimension, you can use the pixel size routine to find a pixel size and grid angle, provided that you first use this command to indicate the mesh size of the grid (mesh openings per inch).  The mesh size for the standard replica grating is 400.  You will probably need to reduce the binned size to 512 with the Set Binned Size command.  When you are done calibrating magnifications where the grid bars are useful, run this mesh size command again and enter 0.  This setting is not retained between runs of the program.

Autofocus command (Calibration - Focus & Tuning sub-menu)

This command will calibrate autofocusing at the current magnification in TEM mode. 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 on a Thermo/FEI scopes, or if a JEOL scope has good RotationAndPixel properties plus image shift calibrations, there is no need to have an autofocus calibration at more than one or two magnifications. It is better to have a few high quality focus calibration over a large focus range rather than poorer calibrations at many magnifications.

As of SerialEM 3.9, 10/11/20, a calibration done in low magnification will be used when autofocusing in LM, instead of automatically going to a standard focus.  You will probably want to calibrate over a much larger range of focus values than the default to get sizeable changes in the shifts during the calibration.

In STEM mode, this command is used to get an estimate of the amount that the beam increases in size as defocus is changed away from focus.  The value is needed for autofocusing, but need not be very accurate.  Nevertheless, if the program eliminates points from the fit and/or suggests that you rerun the procedure with a different focus range, do so.  The focus range and number of focus levels to test are specified in dialog boxes when you start the command.

Set Focus Range command (Calibration - Focus & Tuning sub-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 four 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, or will offset the whole range from the current defocus if a non-zero offset is entered in the fourth box.
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.
4. Offset to apply to the whole range to be tested (negative for underfocus).

For Falcon 2 and 3 cameras, the standard range generally runs into problems with the Falcon Dose Protector, usually just at the overfocus levels.  The easiest solution is to offset the range with the fourth entry.  However, unless there is a reason to measure at higher underfocus than normally, the range should be restricted as well as the offset.  The program will suggest values for the first and fourth entries that will restrict the range on the overfocus side only.

Extended Autofocus command (Calibration - Focus & Tuning sub-menu)

This command will calibrate autofocus over a wider range of defocuses, doing the same defocus steps within the range used for the regular calibration, but calibrating at a sparser interval outside that range and with smaller beam tilt to avoid excessively large image displacements.  The calibration is particularly useful when autofocusing with the View area in Low Dose mode using a sizeable View defocus offset or when using the View area for doing the Eucentricity by Focus task even without a large offset.  It would also allow one to take a calibration at higher magnification with much finer spacing than the default near focus, but the same or larger range than usual.  The full range and interval outside the regular range are set with the next command and have defaults of -300 to 100 microns at a 20 micron spacing.  The command should be run outside of Low Dose mode, with the magnification set at the desired level (e.g., the typical View area magnification in Low Dose mode).  You need to make sure that the program can take usable Focus images at the low and high end of the defocus range.  The actual interval in the extended range will be somewhat less than the specified interval in order to fill each end of the range with equally spaced measurements.

To prevent image displacements from becoming too large to measure at the most extreme defocuses, use full-field images as well as a smaller beam tilt in the extended region.  If calibrating a range of about +/-100 microns at higher magnification, you may need to reduce the beam tilt by more than the default amount.  This is less likely to be needed when doing a calibration over a wider range to be used in View, because the field of view is much larger.

Set Extended Range command (Calibration - Focus & Tuning sub-menu)

This command lets you set the lower and upper defocus limits, the approximate defocus interval, and the reduction in beam tilt to use in the Extended Autofocus calibration outside the regular range.  Four entry boxes will appear for the four parameters.  The lower, underfocus limit should be a negative value.  It should be set to the lower of two values:

1. The most negative View defocus offset that might be used for autofocusing in general, and
2. The most negative View defocus offset that might be used in the Eucentricity by Focus task, minus the biggest Z changes that would typically occur when using this calibration in that task.

Thus, the default value of -300 would cover both general autofocusing with an offset of -300, and autofocusing in the eucentricity task with an offset of -200 and Z changes up to about 100.  Note that it is recommended that the eucentricity task not be done with such high defocus values if a less negative one can be used reliably.

The upper, overfocus limit should be positive and set to cover the range of Z changes that might need to be found in the Eucentricity by Focus task if there is no sizeable defocus offset.  The default value of 100 should be adequate.

The interval has a default of 20 and the program will insist that it be at least 6 microns, the default for the regular range.  The beam tilt reduction has a default of 0.5 and is constrained to between 0.1 and 1.

Standard Focus command (Calibration - Focus & Tuning sub-menu)

This command will store a standard or absolute focus value that can be reapplied in various situations.  In low magnification, use the command to record the current defocus as a standard focus that will be used when calibrating image or stage shift or taking montages. You can calibrate the standard LM focus at one magnification if that is sufficient, or you can do the calibration at as many magnifications as are necessary. When the standard focus is needed at an uncalibrated low magnification, the program will take the calibration from the nearest calibrated LM one, or just assume a value of 0 on a Thermo/FEI scope if there are no calibrations in LM. The script command 'SetEucentricFocus' can also be used to set the focus in this way when in LM.

If not in LM, use this command to store a eucentric focus value for the current magnification.  The script command 'SetEucentricFocus' can be used to return to this focus value, and again it will use the value from the nearest nonLM mag if there is not one at the current mag.  However, on a Thermo/FEI scope, a warning will appear in the log when this substitution occurs.

 If you need to replace existing calibrations, see the entries for 'StandardLowMagFocus' near the end of the SerialEMcalibrations.txt file to determine which magnifications were calibrated previously.

STEM Focus Vs. Z command (Calibration - Focus & Tuning sub-menu)

Use this command to calibrate the relationship between defocus change in STEM mode and a change in physical Z height.  On the JEOL the relationship is close to linear and you will use the output to set a value for the property STEMdefocusToDeltaZ.  On a Thermo/FEI scope, the relationship is nonlinear and may even be specific to the spot size, so the curve from this routine will be stored as a calibration.  The program will step through a range in Z, autofocusing at each step, and smooth the curve at the end by averaging over successive sets of three measurements. You need to have a specimen and imaging conditions under which autofocusing works well.  You should also make sure the stage is at the eucentric height.  When you select the command, you will make entries to two dialog boxes:

1. Total Z range over which to measure focus.  The program will step from half this range below the current Z to half this range above 1.
2. Number of Z levels to measure focus at.

When the routine is done, it reports the slope over the whole set of smoothed points, and over the lower, middle and upper halves.  For a JEOL, the slope over the whole curve might be the best value to use for STEMdefocusToDeltaZ.  For a Thermo/FEI scope, you should also enter this property, using the slope over the middle of the curve.

Base Focus Values command (Calibration - Focus & Tuning sub-menu)

On the Hitachi HT7700, use this command to record the objective lens values at each magnification in HR and HC modes, and the intermediate 1 lens value at each magnification in LM mode.  When changing between magnifications, the microscope changes the focus lens value by a certain amount; this calibration is needed so that SerialEM can subtract off such changes and show a constant defocus when magnification changes.

Astigmatism by CTF command (Calibration - Focus & Tuning sub-menu)

Use this command to obtain a calibration for correcting astigmatism by CTF fitting to images with Thon rings.  The program will measure the amount of astigmatism induced by setting the X and Y objective stigmators to positive and negative values.  The images used for calibration should satisfy the various criteria described in Autotuning by CTF Fitting.  The calibration should be done at a fairly high magnification for more accurate fitting (e.g., pixel size around 0.25 nm).  The defocus should be -1.5 to -2.5 microns.

The stigmator change should be set so as to get an astigmatism value of ~0.8 microns from the CTF fitting.  The easiest way to determine this is just to run the calibration and see how big the 'astig' value is after the first fit with stigmation applied.   Then use the Setup Astigmatism command to change the stigmator value to achieve the desired astigmatism instead.  If the calibration gives astigmatism that cannot be fit (a non-elliptical pattern) then use that command to reduce the value by a factor of ~3.  The default is 0.04 as of SerialEM 3.7.6.   These values have been found useful for Thermo/FEI scopes: 0.1 for Tecnais, 0.3 for Krios, 0.3 for a Glacios, 0.5 for a Talos 120C. Once an appropriate value is determined, the default value can be set with the second entry to the property 'AstigmatismParams'.

Astigmatism by BTID command (Calibration - Focus & Tuning sub-menu)

Use this command to obtain a calibration for correcting astigmatism by beam-tilt induced displacements.  The program will measure the relationship between settings of the objective stigmator and the amount of shift in the image induced by beam tilt in two different directions.  It also needs to measure these shifts for a known focus change.  It starts by measuring the current defocus, then it measures the beam-tilt induced shift at focus levels above and below a middle focus value.  It returns to the middle focus value and measures beam-tilt induced shift for positive and negative changes in the X and Y stigmators.  Matrices are computed from these measurements.

Since the measurements are similar to what occurs with autofocusing, the Focus parameters are used.  There should be a specimen with some features in it; the beam intensity and exposure should be high enough to give clean images and accurate autofocusing.

Several parameters need to be in the right range to get an accurate calibration:

1. Magnification should be high enough to get substantial shifts.  The unbinned pixel size should be at most 0.5 nm for phosphor-based cameras at 200-300 KV, or at most 1 nm otherwise (< 200 KV or direct detectors).  You may want to calibrate at a magnification where this is the case, plus one about 3 times higher.  Focus images can be binned by 4 for phosphor-based cameras at 200-300 KV or by 2 otherwise; results may be slightly more accurate with half that binning.
2. Beam tilt for the calibration should be about twice that used for autofocusing, or about 3 milliradians.  This may require removal of the objective aperture.  It is possible to use a smaller beam tilt for astigmatism correction; e.g., one the same as for autofocusing.  Beam tilt is set with the Set Astig Beam Tilt command in the Focus menu.  On non-Thermo/FEI scopes, the entry box for beam tilt will report the scaling from percent of full-scale beam tilt to milliradians, estimated from focus calibrations.  Divide the desired beam tilt by the scaling and round to the nearest 0.1 to get the percentage value to enter.
3. The amount to change the stigmators needs to be large enough to get sizable differences in beam-tilt induced shift, but not unrealistically large.  See the section above for values that should work on  some Thermo/FEI scopes.  On non-Thermo/FEI scopes, or if you want to check the effect of a value on a Thermo/FEI scope, use the following script:
   ReportObjectiveStigmator
   stigx = $repVal1
   stigy = $repVal2
   newx = $stigx + 0.1
   SetObjectiveStigmator $newx $stigy
   Delay 2
   F
   SetObjectiveStigmator $stigx $stigy
   
   For this to work, you will need a specimen and conditions that give good Thon rings in an FFT.  Autofocus and go to 1 to 2 microns underfocus, then run the script.  Compute the FFT and click at the first minimum along the minor axis of the ellipse to measure the maximum defocus, and along the major axis to measure the minimum defocus.  The difference should be 0.7 - 1 microns.  (If you have the option for CTF fitting turned on, click at the first minimum midway between the axes and use the 'astig' value that it reports.)  Adjust the value '0.1' in the script to achieve this.

Calibrations are stored separately for different magnifications, probe mode on a Thermo/FEI, or alpha on a JEOL.  The Correct Astigmatism routine will chose a calibration from the nearest magnification with a matching probe mode or alpha.

After determining beam tilt or stigmator values different from the default, add a property AstigmatismParams with those values; the beam tilt will then become the default for other users unless they set their own values.

The first time that an astigmatism calibration is run on a particular scope, the adequacy of the delay after changing astigmatism should be checked.  Run Calibrate - Set Debug Output and enter c, then run the astigmatism calibration at the highest magnification that you plan to calibrate.  The shift and drift in unbinned pixels will be reported for each measurement of beam-tilt induced shift, along with the 'operation' index after 'oper' and direction index atfer 'dir'.  Inadequate delay will show up as higher drift for direction 0 on operations 2, 3, 4, and 5.  If this is the case, add a property AstigmatismComaDelays with a delay higher than the default of 900 msec and try again. A more unlikely problem is inadequate delay after changing focus, which will show up as higher drift for direction 0 on operations 0, 1 and 2.  A second value on the AstigmatismComaDelays property can be used to set a delay higher than the default of 500 msec.

Setup Astigmatism command (Calibration - Focus & Tuning sub-menu))

Use this command to set some of the parameters for calibrating astigmatism.  The program will ask for the amount to change the objective stigmators, the middle focus at which to vary astigmatism, and the total focus range over which to measure the relationship between focus and beam-tilt induced image shift.  The first value is important, as discussed for the previous command, but the defaults for the others are probably fine.

BTID Coma-free Alignment command (Calibration - Focus & Tuning - Old Coma-free Alignment sub-menu)

Use this command to obtain a calibration for running the Coma-free Alignment by BTID routine, which will adjust beam tilt to near zero by measuring beam-tilt induced displacements.  This method should no longer be used; it has been superceded by the more accurate Coma-free Alignment by CTF routine.

 The calibration involves measuring the difference between beam-tilt induced displacements at various beam tilt misalignments away from the current setting.  From differences at negative and positive misalignments, it computes a matrix based on differences of these differences.  It is thus particularly important for the displacements to be measured accurately, which means this procedure needs to be done with a large beam tilt and at fairly high magnification.  Another important point is that the coma-free alignment must use displacements with the same beam tilt as for a calibration.  With regard to the parameter settings:

• The maximum beam tilt needs to be at least 10 milliradians; ~15 may give better results; higher than this may be counterproductive.  This beam tilt is set  with the Set Coma Beam Tilt command in the Focus menu.  If there are existing calibrations, the entry box will show their beam tilt values, so that one can pick an existing value.  Otherwise, on non-Thermo/FEI scopes, the entry box will report the scaling from percent of full-scale beam tilt to milliradians, estimated from focus calibrations.  Divide the desired beam tilt by the scaling and round to the nearest 0.1 to get the percentage value to enter.
• The magnification needs to be high enough so that the unbinned pixel size is no more than 0.15 nm for phosphor-based cameras at 200-300 KV, or no more than 0.3 nm otherwise (< 200 KV, or direct detectors).

As for astigmatism calibration, the Focus parameters are used.  There should be a specimen with some features in it; the beam intensity and exposure should be high enough to give clean images and accurate autofocusing.

The objective aperture needs to be removed for the calibration procedure.  The Coma-free Alignment routine will use only half the maximum beam tilt reached in the calibration, so it may be possible to run that with the objective aperture in, if it is large enough and well-centered.  Calibrations can be stored with different beam tilts even at the same magnification, if there is a need to run the alignment with different beam tilts in different situations.  Calibrations are stored separately for different magnifications, probe mode on a Thermo/FEI, or alpha on a JEOL.  The Coma-free Alignment routine will chose a calibration from the nearest magnification with a matching beam tilt and matching probe mode or alpha.

After determining a beam tilt different from the default, add a property ComaParams with that value; the beam tilt will then become the default for other users unless they set their own values.

The first time that a coma-free calibration is run on a particular scope, the adequacy of the delay after changing beam tilt should be checked.  Run Calibrate - Set Debug Output and enter c, then run the coma-free calibration at the highest magnification that you plan to calibrate.  The shift and drift in unbinned pixels will be reported for each measurement of beam-tilt induced shift, along with an 'operation' index after 'oper'.  Inadequate delay will show up as high drift values, particularly for the operations that involve the greatest initial beam tilt (0, 4, 5, and 7).   If drift is high, the delay can be increased from its default of 500 msec with the third value in the property AstigmatismComaDelays (if you have to add the property, use the defaults for the first two delays).

To assess the precision of the calibration, you should run it twice under the same conditions.  The program will always report the mean and maximum difference between a previous and new calibration, averaged over the 8 matrix terms of the calibration and expressed as a percentage of the largest term.  It is probably best to get a mean and maximum of 5 and 10%, respectively, or less.  The two ways to reduce these errors are to increase the magnification and the beam tilt; the latter will have a bigger effect.

Setup BTID Coma-free command (Calibration - Focus & Tuning - Old Coma-free Alignment sub-menu))

Use this command to set the defocus at which the coma-free calibration and alignment will run.

List Calibrations command (Calibration - Focus & Tuning sub-menu)

Use this command to list all focus calibrations by magnification and camera, plus astigmatism and coma-free calibrations with their conditions.

Illuminated Area Limits command (Calibration - Beam & Spot sub-menu)

Use this command to calibrate the limits of the illuminated area readout from the microscope at each spot size and in nanoprobe and microprobe.  Recording these values is designed to protect the validity of calibrations involving beam intensity when the microscope alignment values that determine the relationship between underlying lens values and illuminated area readout change.  These limits, or the single pair of limits provided by the 'IlluminatedAreaLimits' property, determine the scaling between illuminated area and the intensities that are stored intenally.  The first time that these limits are calibrated, the intensity values stored in various calibrations and some user settings are scaled for the change in the limits from the property value, so that these intensities will map to the same illuminated area.  If the limits change after that and the calibration is run again, it will restore the proper mapping of values without any need to scale internal intensity values.  This procedure is based on the assumption that such a change in the limits just reflects a change in scaling between lens values and the readout, not a change in the relationship between lens values and the actual beam size.

If you find that the illuminated area readout from the scope is off from the true beam size by enough that it needs to be fixed and this calibration has not been done, run this calibration first to record the current values. Then get the readout fixed and run this calibration again.

The calibration must be run with the largest condenser aperture in because the illuminated area readout is scaled for aperture size.

Beam Crossover command (Calibration - Beam & Spot sub-menu)

This command allows a calibration of the condenser lens setting for beam crossover at each spot size. This calibration is needed in order to maintain beam intensity calibrations on both sides of crossover. In addition, this calibration should be done before the beam and spot intensity calibrations because the crossover value is stored when those calibrations are done. If the beam crossover changes, which can occur as a result of some alignment procedures on Thermo/FEI scopes, then rerunning this calibration will adjust the C2 settings appropriately in the beam and spot calibrations. First make sure you are at eucentric focus. Go to a high magnification where crossover can be visualized easily. After you select the command, the program will cycle though the spot sizes and ask you to bring the beam to crossover at each.

For a Titan microscope, you will be asked to enter the size of the C2 aperture after running this command.

Beam Intensity command (Calibration - Beam & Spot sub-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. The Beam Crossover calibration should be up to date before this calibration is done.  The calibration must be done outside Low Dose mode.  The calibration is specific to the side of crossover on which it is run, and so would need to be run on both sides of crossover if you plan to work on both sides.

To prepare a calibration, start with the brightest beam that anyone might need calibrations for. For example, 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. Another reason to start at a higher magnification is if the beam is not very uniform; for a non-FEG microscope, you are probably better off spreading the beam at least as large as the screen. Select this command to start the procedure, then check that the beam is still centered. (On Thermo/FEI scopes, the C1 lens (spot size) is normalized, which can affect the beam size and position.  Also, on Titan scopes, the beam position may not be right when the flu screen is retracted only once, so it is recommended that you retract and reinsert the screen before centering it, or else center it on a live image from a camera). 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 the latter is more than 1 a lot of times, or if the calibration fails, the first recourse is to increase the property BeamCalSpacingFactor to 1.2.  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

If you have a K2/K3 camera, the preferred option is to do intensity calibrations on a different camera.  If you must use the K2/K3, then at least the higher spot sizes may require a change of properties to the following:
   BeamCalMinExposure       0.1
   BeamCalMaxExposure      0.4
   BeamCalMinCounts          10000
   BeamCalMaxCounts         30000
   BeamCalInitialIncrement   0.0002
   BeamCalSpacingFactor    1.2
As of SerialEM 3.7, the first four are set automatically, and the property values are ignored.  In addition, this version will use counting mode when the starting dose rate is below 20 electrons/unbinned pixel/sec (or 75 on a K3 camera).

For the weakest spots, you must be sure to condense the beam to be tight around the field of view of the camera before starting, so that you are working with as bright a beam as possible when starting.  The problem with the K2 (and the K3 to lesser extent) is that the noise becomes too big once the beam gets weak, so the procedure has to be run in a way that keeps dropping the magnification instead of making the beam much weaker.  Using counting mode where possible alleviates this problem.

For a Ceta camera, noise is also a problem when the counts get low. Modify the properties to the following, adding the last one to make the calibration routine behave as it does for a K2/K3.  These values have worked in the past, but recently (as of June 2020) a few non Ceta-D cameras have needed the values shown for Ceta-D.  The general procedure would thus be to check the saturation level of the camera; if it is around 6000, use the values for Ceta-D; if it is some other value under 35000, use 85% of saturation for the 'BeamCalMaxCounts' and 10% for 'BeamCalMinCounts'.
   BeamCalMinExposure           0.1
   BeamCalMaxExposure          3.0
   BeamCalMinCounts              2000         (use 500 for Ceta-D)
   BeamCalMaxCounts             30000        (use 5000 for Ceta-D)
   BeamCalInitialIncrement       0.0002
   BeamCalSpacingFactor        1.2
   BeamCalFavorMagChanges  1

These settings will also work for a Falcon 4 camera, with BeamCalMinCounts set to 1000.

For a Titan microscope, you will be asked to enter the size of the C2 aperture after running this command.  SerialEM uses the illuminated area readout as an intensity measure, and the microscope scales this readout by the aperture size.  Thus, intensity calibrations are valid only if the program knows both what aperture size the calibration was obtained at, and what the current aperture size is. Use the Set Aperture Size command to inform the program when the C2 aperture has changed.

If you have existing calibrations from before this feature was added to keep track of the aperture, you can edit the SerialEMcalibrations.txt file to insert the aperture size if you know it.  Look for each line starting with 'BeamIntensityTable'.  If the line has 5 numbers then the mag, you can insert the aperture size (in microns, without a decimal point) just before the mag.  If the line has 6 numbers then the mag and the last one before the mag is 0, you can replace the 0 with the aperture size.  Also, if you know the aperture size at which the crossover calibration was done and there is no line with 'CrossCalAtC2Aperture', you should add a line with 'CrossCalAtC2Aperture' followed by the aperture size.  Finally, if you know the aperture with which the spot intensity calibration was done and there is no line with 'SpotCalAtC2Aperture', you should add a line with 'SpotCalAtC2Aperture' followed by the aperture size listed twice, once for each side of crossover.

Spot Intensity command (Calibration - Beam & Spot sub-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 calibration is specific to the side of crossover on which it is run, and so would need to be run on both sides of crossover if you need to transfer dose information on both sides. The procedure will take pictures with the Trial parameter set, starting at the brightest spot that you choose and working down to the dimmest spot that you specify, 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 the brightest spot to be calibrated and set the Trial exposure so that it gives counts about 1/2 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 a Thermo/FEI scope, 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.

With a K2/K3 camera, again, use a different camera instead if possible.  Otherwise, set the Trial parameters for an unbinned exposure in Linear mode.  The procedure should work well enough if there is still ~1 electron per unbinned pixel per second in an image with the dimmest spot, and if the intensity setting needed for this does not saturate the camera on spot 1.  Try a 1 second exposure.   If possible, verify with a specimen in place that an image with ~20,000 counts (~40,000 with no division by 2) on spot 1 does not get saturated.  Then set the beam for that many counts with no specimen in place and see if it works down to the dimmest spot.  Run the procedure.

For a Titan microscope, you will be asked to enter the size of the C2 aperture after running this command.

Set C2 Factors command (Calibration - Beam & Spot sub-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 Microscope User Interface on Thermo/FEI scopes not using illuminated area for intensity. 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 scope. First be sure that you have a C2 lens readout in your MUI workspace. Then activate the command and type in the value shown in the C2 readout for each spot size.

Beam Shift command (Calibration - Beam & Spot sub-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 camera field. Take a Trial exposure to verify this, and adjust exposure time or spot size to prevent camera saturation.   (If the beam is still too bright, use a smaller condenser aperture, if possible.)  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.

On all microscopes, the beam shift calibration is stored separately for LM and non-LM modes.  On the JEOL, calibrations are stored separately for GIF mode, for different magnification ranges separated by either image shift or beam shift boundaries, and for different alpha values.  When PLA is being used for image shift, every magnification is an image shift boundary.

Refine Beam Shift Cal command (Calibration - Beam & Spot sub-menu)

This command will refine a beam shift calibration on JEOL and Hitachi microscopes to be more accurate over larger distances.  It does this by applying image shift over a large distance and measuring how much the beam moved on the camera due to the inaccuracy of the existing beam shift calibration.  Specifically, the image shift equal to one camera field is applied in four directions to get an initial refinement; then image shift equal to your specified maximum distance in microns is applied in four directions to refine this calibration; finally the maximum shift is repeated both to show how good the calibration would be after the first two refinements and to obtain another refinement.  If the beam stays close to stationary on that last round, then you can be sure that the final calibration is as good as that or even better.  If the beam still moves substantially, the only way to tell how good the final calibration is would be to repeat the whole procedure, using this result as the starting point.

The center of the beam can be detected in one of two ways: by the centroid of the image after subtracting a background, as in the original shift calibration, or by fitting a circle to the edge of the beam, as in beam autocentering, which requires a sharp beam edge as obtained with a FEG.  1) When using the centroid, the beam can be somewhat larger than for the beam shift calibration, up to 3/4 of the size of the field.  If the beam is not entirely in the image for some of the shots, this will reduce the accuracy for that round of 4 shots, but may not matter as long as it does not happen in the final round.  2) When using the beam edge, the most accurate results will be obtained with nearly all of the beam edge in the image.  In principle, the beam can even be larger than the field if it is shifted so that about half of the edge is in the field, and you may get acceptable results with such a beam.  However, if the beam is not round or becomes distorted at high image shift values, a partial edge will give variable center estimates, so it is best to have the whole beam in the field in the initial image.  The center position, radius, and error of the fits to a circle are output to the log.  The range of radius values should be less than ~0.5% of the radius, and the error values should be fairly consistent; if not, the result may not be very accurate.

If projector shifts (PLA) are being used for image shift, be careful not to go out of range at higher magnifications.  The allowed image shift is inversely proportional to the magnification.  When image shift goes out of range it is clipped, which will either stop the routine if a message box is set to appear upon clipping, or give a bad result.  Beam shift can also go out of range with no warning or clipping, which will probably give a black image and a bad result.

The calibrations prepared with this command are stored with a flag that allows them to coexist with other calibrations in the same magnification range (see previous command).

This command is enabled for non-Thermo/FEI scopes provided that a beam shift calibration, image shift to specimen matrix, and camera to image shift matrix exist or can be derived for the current magnification.

Alpha Beam Shifts command (Calibration - Beam & Spot sub-menu)

Use this command to calibrate how much beam shift and beam tilt needs to be changed when going between two low dose areas with different alpha settings on a JEOL.  The program will go to a series of alpha settings, starting with the lowest, and ask you to center the beam at each alpha, and adjust the beam tilt if desired.  First, you specify the number of alpha values to calibrate.  If you enter 0, this will wipe out any existing calibrations.  Adjust the beam at each alpha setting, and press OK.  If you press Cancel, beam shifts and tilts will be saved up to but excluding the current alpha setting.  You do not need to be in Low Dose mode to run this procedure.

When going between two Low Dose modes with different alpha settings, the difference in calibrated beam shift and tilt between those two alphas will be applied.  Small remaining beam shifts can then be compensated using the beam offset feature in Low Dose mode.  Additional small beam tilts can be compensated as well if the property 'LowDoseBeamTiltShifts' is set.

These calibrated changes will be applied a similar fashion in two situations when using the Navigator: 1) when using Realign to Item in the Navigator to realign to a map originally taken in low dose mode, and 2) when taking an Anchor map where the map marked as "For anchor state" was taken in low dose mode.

Spot Beam Shifts command (Calibration - Beam & Spot sub-menu)

Use this command to calibrate the beam shifts needed to keep the beam centered when changing spots.  This calibration is essential on the Hitachi HT7700 and it might be useful elsewhere.  For Hitachi, this calibrations has to be done separately for HC and HR modes; on a JEOL, it has to be done separately for MAG and SAMAG modes if SAMAG is defined as a secondary magnification mode.  Thus, when you run the calibration, it will be done for the current mode of the microscope. The program will proceed from dim to bright spots, starting at the spot number that you specify after selecting the command.  At each spot, it will ask you to confirm that you have centered the beam.  You need not go all the way to the brightest spot; if you press Cancel instead of OK, it will not record a shift for the current spot size.  Any existing calibrations will be lost; calibrations will be stored only between the starting and ending spot.  When changing spot size between two sizes and a calibration exists at both, the program will apply the difference in recorded beam shifts to correct the beam position after the spot change.

List Calibrations command (Calibration - Beam & Spot sub-menu)

Use this command to list some spot and beam related calibrations.

Set Aperture Size command (Calibration menu)

Use this command to inform the program of the current size of the C2 aperture on a Titan microscope, where the illuminated area read from the microscope and used for intensity is scaled by this aperture size when the aperture is changed.  The program will scale the intensity values in the beam intensity, spot intensity, and crossover calibrations so that they are valid for this aperture size.   Once the aperture size is set either with this command or  after one of those calibrations, it will be remembered between sessions in the short term calibration file.

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 1/2 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.

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 a Thermo/FEI scope (or a 0 for a JEOL). These numbers are listed in the form required for the magnification table in the SerialEMProperties.txt file. Different numbers will be output in EFTEM mode than in normal lens mode. On a Thermo/FEI scope in EFTEM mode, the normal mode magnification and rotation will be printed on each line before the EFTEM values, so that the full table can be pasted directly into the properties file.  However, make sure that the table entries for normal mode are correct before adding the EFTEM values.  If necessary, correct the table for normal mode and add the screen down magnifications (if they differ), then restart SerialEM with the corrected properties file.  Then run this command in EFTEM mode to get the EFTEM table combined with the correct one for normal mode.

On a JEOL, the magnification table is followed by a table of camera lengths, which is needed for the program to interpret diffraction mode correctly. This table should be cut and pasted into the properties file, with the first line being 'CameraLengthTable nn', where nn is the number of camera lengths listed.  On a JEOL, if magnifications in low mag mode are disabled above a certain point, be sure to add the property 'HighestLMindexToScan' with the number of remaining enabled LM mags before running this routine.

In STEM mode, on a JEOL, the program will list all of the STEM magnifications and produce a table that can be inserted into the Properties file, plus an entry for LowestSTEMnonLMmag.  On a Thermo/FEI, STEM magnifications are not identified by index values, so it is necessary to step through the magnifications with the knob on the microscope.  The program will tell you to enable "LM scan" in the microscope STEM interface, and start at the lowest magnification that is reached with that enabled.    You will need to step through the magnifications twice, first in nanoprobe then in microprobe mode.  Be very careful not to turn the knob again until the program has printed a new magnification in the log. At the end, the program will output the table, two entries for LowestSTEMnonLMmag, and an entry for LowestMicroSTEMmag.  You may find that some magnifications show up in both LM and nonLM.  The program has no good way to distinguish these and they will show up as "S?" in the microscope status panel.  This may not be a problem in practice.  One solution would be to skip the LM magnifications, but a better solution would be to go through a microscope alignment that sets the STEM magnifications and set slightly different values that avoid the duplications.  Have this done before doing any calibrations, and get a new new listing of the magnifications.

On a Thermo/FEI scope, this routine will not produce a camera length table.  The only purpose for such a table is to allow the program to display '->' and the camera length to be changed to when using the magnification/camera length spinner in the  Microscope Control panel.  If you plan to use this panel in diffraction mode, you can add a table prepared with the following script:

SuppressReports
lastlen = 0
num = 0
firstEnd = 0
secondEnd = 0
loop 60 ind
   SetCamLenIndex $ind
   ReportCameraLength
   len = round ($repVal1 * 1000 0)
   if $len > $lastlen
      EchoEval $ind $len
      num = $num + 1
   ElseIf $firstEnd == 0
      firstEnd = $num
   ElseIf $secondEnd == 0
      secondEnd = $num - $firstEnd
   Endif
   lastLen = $len
EndLoop
echo NumberOfCameraLengths $firstEnd $secondEnd

Neutral IS Values command (Calibration menu)

On a JEOL, this command will cycle through the magnifications and measure the neutral settings for image shift (or projector shift when using that 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.  This command needs to be rerun after a service alignment that may have changed the values.  Before running the calibration, make sure that the microscope alignment panel is set to User mode instead of Maintenance mode.

On a Hitachi HT7700, this command needs to be run to record the IA deflector shifts in LM mode, and either the IA or PA deflectors in HR mode, depending on which is being used by SerialEM for image shift there.  (This is determined by the property HitachiPAforHRmodeIS.)  There are no fixed alignment values for these deflectors; the scope just restores the value last seen at a magnification when it returns to that magnification.  Thus, before running this command, you must make sure that the magnifications are reasonably well aligned at least in LM mode.  Do this without SerialEM running, to ensure that no extraneous changes are imposed by SerialEM as well.  You should do the same in HR mode if the same deflectors are being used to align those mags as for image shift in SerialEM. If, instead, the IA deflectors are used by SerialEM for image shift and the magnifications are aligned with PA, there is no conflict, but you should step through the HR magnifications and make sure that IA is zeroed out.  After the current status of the deflectors has been checked, run this command to record all of their values.

Restore Neutral IS command (Calibration menu)

On a Hitachi HT7700, use this command to restore the deflectors used for image shift in LM and HR modes to the values recorded with the Neutral IS Values command.  This is useful if you find the magnifications misaligned, or if SerialEM has left the deflectors in an inappropriate state.

Stage Stretch command (Calibration menu)

Use this command to save the most recently applied transformation of Navigator points into a new registration in the calibrations. The transformation can then be used to estimate the stretch in the stage coordinate system. Such an estimate allows greater accuracy when transforming Navigator points using fewer than 5 registration points, or after aligning rotated images in the Align with Rotation dialog .

This command is available only if the transformation was based on a fit to at least 5 registration points and contains a rotation between 70 and 110 degrees. To obtain such a transformation, go to widely separated stage positions, center a recognizable feature on the camera, and add either the stage position or a map at each location to the Navigator table. If possible, you should mark at least 6 registration points, as that will give a more robust transformation than 5. Rotate the specimen by 90 degrees, find the locations again and add a Navigator item at each. Mark them as registration points with the corresponding values, then select the Transform Items command in the Navigator menu. The error in the fit should be only a few microns, if possible. Then select this command. The program will tell you the underlying stretch implied by the transformation, and also the mean magnification change contained in the transformation. The latter should be very close to 1 (perhaps within 0.0005 of 1) and the stretch itself should be plausible (perhaps between 0.95 and 1.05). The transformation will be stored after you confirm that you want to; remember to save calibrations.

High-Defocus Mag command (Calibration menu)

Use this command to measure the change in magnification and rotation between an image taken near zero focus and one at a high negative defocus.  The program can use such measurements to adjust the stage calibration when using View or Search images in Low Dose mode with a large defocus offset.  Montaging and Navigator operations can then operate accurately with such images.

Using a View defocus of -100 microns or more can change the magnification by 5-20% and rotate images by 2-10 degrees.  The rotation is proportional to defocus but the magnification change is nonlinear, changing faster at higher defocus.  Such changes are enough to make the alignment of montage pieces and the navigation to selected stage positions unreliable.  One solution to this problem is to do the stage calibration for the View magnification at the desired defocus.  However, this solution is inflexible, as it applies only to that defocus and may also apply only to a narrow range of beam intensities (at least on Tecnais, the magnification change depends strongly on the C2 setting).  The calibration provided here avoids these problems.  The magnification change and rotation can be measured at several defocus levels and a range of intensity values.  The program can then interpolate among these measurements to compute a good stage calibration anywhere within this range of conditions.  Once you get set up to run this calibration, it is relatively easy and quick to run it for the different conditions.

The Beam Crossover calibration should be done before this calibration so that differences between intensities can be assessed properly.

To do the calibration, you need a specimen with enough image detail for good correlations, and also enough exposure to give reliable correlations.  A specimen with periodic features is acceptable as long as there is not much shift between the near-focus and defocused images.  The procedure is this:

1. Find an area with good non-periodic image detail.
2. Adjust the beam to the mid-range of the anticipated intensities to be used.
3. Adjust the exposure time to give a clean enough image for correlations.
4. If you want to reset defocus, do it before you take the first image to be used in the procedure.
5. Take an image within a few microns of zero defocus.  If you use View in Low Dose mode, set the defocus offset to 0 and autofocus to the desired focus.
6. Copy the image to the Autoalign buffer, which is shown in the 'Align To' button in the Image Alignment & Focus control panel.
7. Change the focus to the first focus to be calibrated, such as -100 microns.  If you use View in Low Dose, just set the defocus offset.
8. Take an image.  You may need to adjust the exposure time to compensate for changes in intensity.  You may need to shift the image to show approximately the same area (or very nearly the same area if there are periodic features).
9. Find two well-defined points, spaced apart by at least half the width of the image, that are recognizable in each image.  You can use the Insert and Home keys in the 6-key cluster to toggle between these buffers.
10. Draw a line between the two points in each image.  To draw a line, hold down the Shift key, depress the left mouse button with the cursor over one point, drag the mouse to the other point, and release the mouse button.  The program will insist on drawn lines unless you are within 10 microns of an existing calibration or there are already calibrations at higher and lower defocus that it can interpolate between.
11. Run this command.  The program will show instructions, but only once.  After you press 'Yes' to proceed, the program will do a search to refine the scaling and rotation implied by the two lines.  At the end, it will transform the image in A by the scaling and rotation and toggle between the near-focus image and the transformed image.  Then it will ask you if those appeared aligned.
12. Press 'Yes' to accept the calibration, or 'No' to reject it.  If the images were not aligned, examine the lines to see if they can be improved, or increase the exposure time if necessary.
13. Change the beam intensity by a factor of two, or to one end of the range of intensities that is expected to be used.  Take an image and run the command without drawing a line.  The lines are required in each image only if there is not already a calibration at the given defocus level.
14. Repeat step 12 at the other end of the expected intensity range, or at several intensities levels spaced apart by factors of two.
15. Change focus to the next focus level to be calibrated and repeat from step 7, drawing a line between the two points in the new image.  The low-defocus image should still be in the Autoalign buffer with its line still present.
16. Be sure to save the calibrations when done.

On Tecnais, calibrations at -100, -200, -250, and -300 microns would provide sufficiently accurate interpolation for any defocus down to -300.

The calibrations are stored separately for different spot sizes, but the program will simply use calibrations from the nearest spot size rather than requiring one at the current spot size.  The magnification change and rotation do not vary significantly with spot size on Tecnais.  You can start out by calibrating at the brightest spot that is expected to be used for Low Dose mode, and this may well be sufficient.

Calibrations are also stored separately for nanoprobe and microprobe mode on Thermo/FEI scopes, and in this case only calibrations from the current mode are used.

If you need to redo the calibrations, do them at intensities near those in the existing calibrations, and the program will remove each nearby existing calibration.  There will be a message in the log window when this occurs.  The criterion for this removal is set by the property 'HighFocusMinIntensityDiff', which has a default of 0.2 in underlying intensity units.  Use the List Focus-Mag Cals command described next to determine the intensity values to use.

High-Defocus IS command (Calibration menu)

Use this command to calibrate how the image shift calibration changes at high underfocus.  With adequate calibrations, many things will work better when using highly defocused View or Search images in Low Dose mode:

• Shifting an image with the right mouse button or shifting to a marker point with Shift-P or the To Marker button will work accurately.
• Image shift montages will have more accurate overlaps between adjacent pieces, so the piece alignments may work better.
• Setting the View or Search shift offset will work in one or very few iterations.
• When Realign to Item uses image shift to go to the target position from the center of the map frame, it will do so accurately.

These operations make use of both high-defocus image shift and magnification calibrations:

• The specimen position will stay nearly centered when switching between Record and View or Search areas even with a substantial image shift present.
• Setting the View or Search shift offset can be done with images that have a pre-existing image shift, and works independent of applied image shift, thanks to the ability to maintain position when switching between areas.
• Setting the image shift vectors for multiple Records by visualizing positions on a defocused View or Search will be more accurate and might be adequate.
• Reset image shift will be more accurate when the current Low Dose area is View or Search.
• Marking Navigator positions on an image-shifted View or Search image, or displaying positions on such an image, will be more accurate.

To get the calibration, the program will do an image shift calibration at a known defocus and compute the change from the existing calibrated image shift matrix and the measured matrix, expressing this change as a scaling, rotation, and stretch.  The scaling and rotation will be stored; the stretch (basically, the remainder after accounting for rotation and scaling) is just a possible indicator of a problem with the new or old matrix.  The magnification, spot size, intensity, and probe mode on a Thermo/FEI scope are also stored.  However, there is no dependence on magnification, and probably none on spot size.  There is a strong dependence on intensity on Tecnais, and a much weaker one on a Talos.  Thus, the calibration should be done at two or three intensity levels bracketing where it will be needed.  It is probably best to do the calibration at a typical magnification and spot size for the defocused View images; however, it should not be done in Low Dose mode, and will use the Trial camera parameters as a starting point.

The Beam Crossover calibration should be done before this calibration so that differences between intensities can be assessed properly.

 Because the existing image shift calibration is a reference, it is recommended that the standard calibration be redone first, near focus, so that it is up to date. Also, you should set the number of rolling buffers to at least 6 so that you can check the alignments if necessary (set 'Roll Buffers A -> F' in the Buffer Control panel.)  Before starting each high-defocus calibration, the microscope should already be at the desired defocus and intensity setting, and the beam and camera settings should be appropriate for doing image shift calibrations at that defocus.  When you start the calibration, you will be asked what defocus you have set the microscope to.  The first time in a session, the default choice will be -50, but thereafter it should come up with the correct value based on the actual focus readings from previous calibrations.

The image shift calibration runs with only 6 images, 3 in each direction; drift is estimated from the shift between the middle images (the second and fifth) and subtracted from the measured shifts.  The drift and the consistency between shifts is reported in the log.  If the calibration is judged bad enough, you will be asked whether you want to forget it; otherwise the results will be stored as a high-defocus calibration.  You can scroll through the images to check the alignment.

A convenient way to do the calibration interactively at a series of defocus values is

1. Pick an intensity value, magnification, and spot size, and make sure that good images can be obtained with a binned Trial images over the defocus range to be calibrated.
2. Set the focus increment in the Microscope Control panel to 50.
3. Focus.
4. Decrement the defocus by 50
5. Run the calibration, entering -50 for the defocus, and make sure it is good.
6. Repeat steps 4 and 5 until the highest desired defocus is reached; the correct defocus should be offered in the entry box.

If the whole procedure is running smoothly, an even easier way is to do step 1, focus, and run a script like:

curDef = -50
Loop 6
   CalibrateHighFocusIS $curDef -50
   curDef = $curDef - 50
EndLoop 

This will calibrate in 50 micron steps from -50 to -300 and leave the defocus at the high value.

List High-Focus Cals command (Calibration menu)

Use this command to list the calibrations of magnification and rotation for different defocus values done with the High-Defocus Mag and High-Defocus IS commands.  The calibrations are sorted by defocus and intensity, and the list shows the scaling of the magnification and the degrees of rotation for each.

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, or to determine the startup delay for some other types of cameras, as well as the flyback time for a STEM camera. With a proper startup delay, the program can do several microscope operations (image shift, stage shift, magnification change) after the exposure ends for some cameras. Also, an approximately accurate startup delay is needed for the delay after an image shift or other operations to be imposed correctly. The procedure uses full-field images binned to 1024x1024 pixels by default.  For a non-STEM camera, it measures the mean counts in such images 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.

This routine gives unreliable results for both K2 and K3 cameras, but by following the notes below you should be able to get reliable post-actions with a K2.  Post-actions are disabled by default for K3 cameras, and the camera property 'AllowPostActions 1' would have to be added to enable them.  It seemed for a while that a startup delay of 1.5 sec was reliable, until a report came in of problems with a delay of 1.8 sec.  If you want to enable and try post-actions with a K3, add at least 0.7 to the suggested value from this routine and do not use a value less than 1.9 sec.

For a K2 Summit or K3 camera, the procedure takes super-resolution mode images, which have the longest startup time.  The mean counts do not need to be very high, because the mean counts in such images are very reproducible.  The binning does not matter.   

When the operation is started, the program first takes several reference pictures. It then varies the timing of initial blanking to find a duration that will occlude about the first half of the exposure.  Then it takes many pictures (100 by default, or 40 for K2 Summit or K3) after adjusting the timing parameters to give this amount of extra beam blanking; the counts in such pictures indicate how long this blanking occurred during each exposure. From the range of values, the program estimates values for several properties: StartupDelay and ExtraBeamTime for all non-STEM cameras, plus MinimumDriftSettling and possibly BuiltInSettling for a Gatan camera with two shutters.  If the blanking apparently ended before or after the exposure for some of these pictures, the program will recommend rerunning the procedure with a longer exposure time.

For a camera with the NoShutter property set, the program will switch from NoShutter 2 to 1 for the timing measurements.  This means that during the reference images, it will unblank before requesting the exposure and reblank after the image is acquired and returned.  During the test images, it will unblank at various times after the acquisition is requested, but still not reblank until the image is returned.

For a K2 Summit camera but not K3, the procedure starts with a much longer initial blanking to save time.  If this blanking is too long, a shorter value can be set with the general property 'CalibTimingK2InitialStartup'.  Because a startup delay measured in linear or counting mode with earlier versions of SerialEM would be considerably shorter and can lead to problems, the program will not allow post-exposure actions if the StartupDelay property is less than a minimum value.  If your camera gives a startup delay less than this limit, the program will tell you that it is not safe to use and recommend setting the delay above the default minimum.  Although it is possible to add the property 'K2MinStartupDelay' with a number less than your measured delay, experience has shown that this is unreliable.  This property has no effect with a K3, where initial indications are that the startup delay is much shorter and more reliable.

As of SerialEM 3.7, K2 and K3 exposures with dose fractionation will have an added delay of 5 ms per frame by default (set by the camera property 'StartDelayPerFrame').  This addition should give reliable timing even with the default minimum startup delay.  The signs that the startup delay are too short are: when calibrating image shift, images after the first one may be smeared or the calibration may be bad; when taking tilt series, images after the first few tilts may be smeared; when saving frames during tilt series, the final frames of the exposure may have excessive drift within each frame; when using the MultipleRecord script command and saving frames, the frames near the end of the exposure may shift to the next position.  If turning off the Post-actions option in the Camera menu solves the problem, then the StartupDelay is too short.

Saving frames with the MultipleRecords command, taking two off-center shots and a center shot after them, is an efficient way to test whether the startup delay, or possibly the added time per frame, is too short.  Problems will appear in the first two sets of frames.

For a STEM camera, the program does not use the mean counts in an exposure but instead finds the line in the image where the beam was unblanked.  To take interpretable images, it cancels any contrast inversion or image rotation being done in SerialEM.  The default exposure time is 1.2 seconds, which helps to give an accurate estimate of the flyback time (time between each line of exposure).  Fewer test images are needed to get the final estimate of startup delay, so the default there is 30.  At the end, the program will recommend entries for StartupDelay and AddedFlyback. For DigiScan, this should be a small number; it is added to the flyback time set in the DigiScan interface.  For Thermo/FEI STEM, it will be a large and highly variable number, and it is necessary to run the procedure described next to get a more useful set of flyback times.  The program may also recommend an entry for MinimumBlankingTime, which may be needed to get the next procedure to work reliably.

Quick Flyback Time command (Calibration menu)

This command will run the Camera Timing routine in a special rapid mode to estimate the flyback time (time between successive lines) for Thermo/FEI STEM with particular acquisition parameters. This is necessary because the flyback time for Thermo/FEI STEM varies over a large range and somewhat unpredictably, depending on exposure time, binning, subarea size, and to a small degree, on magnification.  An accurate flyback time is needed for dynamic focusing to work optimally. 

When you select the command, the program will ask which camera parameter set to run the timing for.  Answer with the first letter of parameter set name (upper or lower case).  The program will then run the timing routine with either 3 trials at 3 startup delays or 4 trials at 4 startup delays, depending on exposure time.  At the end it will report the flyback time and startup delay and ask if you want to store them in the table of timings, replacing an existing value if there is one.  When the program exits, this table is automatically stored in the file SEMflybackTimes.txt in the SerialEM configuration directory (C:\Program Files\SerialEM or C:\ProgramData\SerialEM).  The table keeps track of the exposure time, binning, image size, magnification, and date/time at which each measurement is made.

The flyback time has a predictable, linear dependence on exposure time over some ranges of time.  However, there can also be a small interval within such a range with values that differ from the linear relationship by 20% or more.  For example, with full-sized images binned to 1K, there are special intervals at 3 and 6 seconds.  Also, the slope of the linear relationship can change between low and high exposure times.  When doing dynamic focus with a given exposure time that is not in the table, the program will attempt to interpolate or extrapolate from entries in the table with nearby exposure times and other conditions matching.  It will even eliminate one outlier when finding the linear relationship between flyback time and exposure time.  This estimate may be adequate over some ranges of exposure times and not good enough in other regions.  You thus have three choices: 1) use dynamic focus only with parameters that have a measured flyback time and do not vary exposure during a tilt series; 2) vary exposure in discrete steps where the flyback time is measured (using the ability to vary parameters like exposure time during a tilt series); or 3) vary exposure continuously over a range where there are no special intervals.  The second and third strategy in particular would require this timing routine to be run many times.  There is a script command, 'QuickFlyback', that will run this routine and save the result automatically in the table for a specified camera parameter set and an exposure time that replaces the time in that set.  For example, a script like this could be used to measure a series of times at two different intervals.

       exptime = 0.6
      interval = 0.2
      loop 15
        QuickFlyback R $exptime
        exptime = $exptime + $interval
      endloop
      interval = 0.5
      loop 10
        QuickFlyback R $exptime
        exptime = $exptime + $interval
      endloop
      

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. It cannot be run for direct detector cameras, which have other constraints that prevent very small exposure times. 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 - 1/2 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.

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 with programs in IMOD. 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.

At high magnification, addition delay time after moving the stage may be needed.  This can be imposed by setting the property 'DistortionStageDelay' to the desired value in milliseconds, which will replace the default delay time set by the property 'StageMoveDelay'.

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.

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.