Acquiring Tilt Series

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

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

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

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

Minimizing tracking errors

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

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

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

Bidirectional tilt series

A different strategy, running the tilt series in both directions from zero tilt, can be used to avoid problems in reaching an initial high tilt angle and in tracking accurately when coming down from a high angle.  This strategy is most useful in two situations, running multiple tilt series with cryo-specimens automatically, or taking tilt series at very high magnifications, but it may also be preferred for single tilt series with many cryo-samples.  Its main disadvantage is that it introduces a discontinuity in the tilt series at the starting angle.  Almost certainly, tilt angles will not be evenly spaced across this discontinuity because of backlash.  Plastic sections will shrink in the first half of the series, causing a substantial size change between the zero-degree image and the starting image of the second half of the series.  On a few microscopes, stage imperfections cause an apparent change in X-axis tilt between the two halves of the series.  All of these problems can be accounted for when aligning the tilt series in IMOD.  In version 4.7, the eTomo user interface allows one to specify that a tilt series is bidirectional and sets some appropriate parameters automatically.   The Scan Header function of Tomogram Setup also detects whether a series from SerialEM is bidirectional and sets the option appropriately, because the header of the output file has an entry in its second title line showing the starting angle after 'bidir = '.

Although bidirectional series avoid some tracking difficulties, they involve a different challenging step, returning to the starting angle for the second half of the series.  The program takes a lower magnification image at the starting angle, the 'bidirectional anchor' image, and aligns to this image after returning to that angle for the second half of the series.   However, this re-alignment occurs after a much bigger tilt angle change and longer time interval than any other alignment operation in a tilt series, so it may require an image with a larger field of view than in other operations.  For example, in Low Dose mode, you may find that View images with a relatively small field of view work in all other aspects of the series but are inadequate for the anchor image.  Because of this vulnerability, the Tilt Series Setup dialog shows the anchor's field of view in microns and highlights the value when a magnification is chosen that makes the value too small.

In order to produce output files in the proper order, SerialEM restacks the data from the first half of the series into a new file, in inverted order, before it adds images from the second half.  This restacking is done in the background while the program is tilting back and realigning to the starting position.  When those operations are done, it then finishes the restacking before tilting to the first angle of the second half.  If a metadata autodoc (.mdoc) file is being written, a new file will also be produced in the proper order.

Dose-symmetric tilt series

Dose-symmetric tilting is designed to maximize the acquisition of useful high-resolution information, with the least beam damage, from the lowest tilt views where the images have the highest quality.  This is often referred to as the 'Hagen scheme', first described in Hagen, Wan, and Briggs, J. Struct Biol. 2017, 197: 191-198.  SerialEM expands on the basic approach there, provides more flexible tilting options than are available with scripting, and implements it in the Tilt Series Controller, so that nearly all the usual features of tilt series acquisition are available (most notably, predictive tracking, stopping, backing up, and resuming).  This tilt scheme is available only in Low Dose mode.  This method has previously been suitable only on well-performing equipment; while the implementation here may expand its applicability somewhat, there is no guarantee that it will produce superior rather than worse overall results when used with less well-behaved equipment.  Whether this tilting method actually helps or hurts should be evaluated in such cases.

Here are some more details on the features of this tilting method.  Additional details can be found in the help for the Dose Symmetric Tilt Series Parameters dialog and for the first section of the Tilt Series Setup dialog.

• Instead of taking a fixed number of tilt steps on each side of the starting angle, the program allows you to define up to three segments of the tilt series: 1) initially alternating between the two sides after a chosen number of consecutive steps; 2) continuing to alternate after increasing the number of consecutive steps on each side by a chosen amount above a specified angle, either once at that angle or repeatedly at an angular interval; 3) ending the alternations at a chosen angle and completing first one side, then the other.  The entries for all these choices are made in the Dose Symmetric Tilt Series Parameters dialog, which includes a line summarizing the angles at which the number of tilt steps change.
• In its original form, dose-symmetric series were not feasible or sensible for side-entry holders, where large tilts radically change the location of nitrogen in the dewar.  The features here may be useful for implementing a strategy that minimizes such large tilts but still provides some of the benefits of dose-symmetric tilting - but see the caution above.
• The program saves various information before switching to the other direction of tilt and restores the corresponding information from the other direction: the image shift and defocus so that the position in X, Y and Z is restored; and intensities and frame times for a direct detector so that any of the methods of changing specimen exposure during the series can be used.  In fact, these value are saved after every tilt so that it is possible to return to any angle.
• The program stores four alignment references: the Record and tracking references for negative tilting in the usual buffers D and E, and Record and tracking references for positive tilting in buffers F and G.  While Record references are saved there on each tilt, the tracking references may not be updated that often if the predictions of specimen position indicate that tracking can be skipped.  If you stop during the series, the program will know what tilt direction is active, and will autoalign a Trial or Record image to the appropriate buffer (the Align To button in the Image Alignment and Focus control panel is always labeled to indicate the appropriate buffer).  In addition to the active references, the program uses most of the rest of the buffers to keep a stack of previous tracking references, which can be moved into place when backing up to a previous angle.
• When either an excessively dark image is acquired or autofocus fails close to the highest tilt angle on one side, the program ends the tilting on that side and continues with tilts on the other side, behaving in many ways like the second half of a bidirectional series.  You can also use Terminate in the Tilt Series menu to end the tilting on one side and finish the other side.  In either case, when the series reaches its designated end, you can use the One Loop button in the Camera & Script Tools panel to extend the series one image at a time, or open the Tilt Series Setup dialog and raise the tilt range on that side of the series, just as for a bidirectional series.  However, if the series reaches its defined end on both sides and stops, the One Loop button cannot be used since the sequence of further tilt angles is not defined; the only option is to open the dialog and extend the range on the side that just reached its end.  (To extend the range on the side that finished first, you would have to back up to the tilt just after the last one on that side, at which point either tilt limit can be extended; but then you would have to reacquire images on the side that finished second.)
• The program assumes that a starting angle other than 0 represents the tilt angle at which the specimen itself is near zero tilt (i.e., it assumes that dose-symmetric tilting around any other angle but zero tilt of the specimen makes no sense).  Thus, all changes in parameters that depend on the absolute value of the tilt angle, such as change of exposure by an inverse cosine to a power or changes imposed in the Tilt Series Scheduled Changes dialog box, are applied based on the current angle minus the starting angle of the series.
• Stage backlash is handled by making sure that the tilt angle is always approached from one direction, namely by applying a backlash correction whenever tilting in the other direction.  By default, the backlash direction is toward the first tilt limit, specified in the Tilt to text box in the Tilt Series Setup dialog, which is the direction of the first tilt.  If one backlash direction is preferable for mechanical reasons, the direction can be specified with an entry in the SerialEMproperties.txt file, 'TSDosymBacklashDir', followed by -1 or 1 for backlash in the negative or positive direction (i.e., backlash correction applied when tilting in the opposite direction).
• Another property setting may be needed or desired.  The Hagen scripts restore the stage position in X and Y after each tilt because this position can change slightly during tilting.  SerialEM will do this by default for series taken with the dose-symmetric tilting, but not other series.  However, restoring stage position was found to give less predictable positions on an old F20.  The default can be overridden by setting the property 'TSRestoreStageXYonTilt' to 0 to prevent the restoration, or 2 to have it occur for all tilt series.

Control of specimen exposure

Because a sample becomes thicker in the direction of the beam when tilted, a smaller fraction of incident electrons reaches the camera at higher tilts.  It is usually desirable to keep the signal-to-noise ratio in the acquired images roughly constant through the tilt series, which requires greater exposure to the specimen at higher tilts.  The default (and original) method for increasing exposure at higher tilts is to increase the beam intensity by condensing the beam.  The motivation for this approach is that beam-induced drift depends primarily on the total current hitting the sample rather than its local flux; condensing the beam thus tends not to increase this component of drift.  However, it is also possible to increase exposure time instead of beam intensity, which is preferable in some cases.  If you choose this approach, it is important to assess whether drift-free images can be obtained with the highest exposure time needed.

Whichever method you choose for increasing specimen exposure, there are two fundamentally different ways of controlling the change.  One is to adjust the intensity or exposure time after each Record image so as to maintain a constant number of counts through the series.  This method is generally suitable for plastic-embedded samples.  The other method is to adjust intensity or exposure time by a formula based on the cosine of the tilt angle.  This method is preferred for cryo-samples because the total dose and its relative distribution through the series can be determined accurately.

Starting a tilt series

When taking a tilt series in one direction, from one extreme angle to the other, the TSC can be started either at a low tilt angle or at the starting tilt angle. You can safely start at low tilt if you are fairly confident that:

• The camera parameters (exposure time and drift settling) are suitable for getting good pictures at high tilt.
• Autofocusing will work well enough to find focus at high tilt.
• Either the beam intensity or the exposure time can be adjusted automatically to give the desired counts in the image without incurring problems: i.e., without the beam becoming too small and encroaching on the camera area, or without a longer exposure time causing excessive drift in the images.

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

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

1. Decide if you are going to start at low tilt or go to high tilt before starting the TSC.
2. If you are going to high tilt before starting the TSC, refine eucentricity. Select the 'Refine and Realign' command if you have already centered your region of interest and plan to use the 'Walk Up' procedure; otherwise 'Fine Eucentricity' is sufficient unless the magnification is high enough that you might lose your selected area.
3. Go to high tilt. Consider using 'Walkup & Anchor' to save a mid-tilt anchor, as described above.
4. Make sure that the exposure settings for Focus, Trial, and Record give images without drift.
5. Do autofocus and see if it gives a well-focused Record image with the best focus in the center of the field. If not, you can use the Move Focus Center command in the Focus menu to move the center of focus to the middle of the field, or you can vary focus by hand. Once focus is found, you should run the Measure Defocus command in the Focus menu and use the reported value to set the target defocus.
6. Set the beam intensity to give the desired level of counts. You can do this by hand or use the Set Intensity command in the Tasks menu. This can be done with either a Trial or a Record image in buffer A. When you select the command, the message in the dialog box will show the mean counts that a Record image would give with the present beam intensity. You can enter either a new number of counts, or a factor by which to change the intensity. If you change the number by much (more than 10-20%), you should take another image and look at the beam on the screen to make sure it is not too small. If you cannot comfortably achieve the desired value, just adjust the beam so it is large enough. You will be able to select an option to reach the desired target number of counts at a lower tilt angle, and the program will reach this target as fast as it can without making the beam smaller than this initial setting.
7. Take a final Trial image and center the features of interest.
8. Open the Tilt Series Setup dialog box in the Tilt Series menu and check the entries from top to bottom, paying particular attention to the following:
9. Check the starting and ending angles and angular increment.
10. Make sure the delay time after tilting is appropriate for the holder and tilt increment (small increments need less delay time; heavy holders need more).
11. Decide whether you need low magnification tracking. This is recommended for magnifications above 50,000, and might be needed at a somewhat lower magnification if you are starting at a particularly high tilt angle (say, above 70 degrees).
12. Make sure the limit on image shift is appropriate for the objective aperture size and other conditions. On the 300 KV Tecnai or Polara, operating at a relatively low magnification, montaging, and using low dose may all require a smaller limit on image shift, perhaps as low as 1 micron. When the limit is reached, the TSC will automatically reset the image shifts, move the stage to compensate, and recover from the physical disturbance caused by this movement.
13. Make entries for beam intensity control. Checking 'Do not increase intensity above value for first saved image' is almost always a good idea. If you are starting at high tilt and needed to set the beam to give less than the desired target number of counts, check 'Keep intensity below current value (use if intensity already set up)'. This will achieve that target as soon as possible without overconstricting the beam. On the other hand, if you are starting at zero tilt and know that you need to reduce the number of counts at high tilt, fill in the desired target number in the 'Set intensity to keep mean counts of Record images at' text box, check the 'Taper counts down to' option, and put your reduced target for the highest tilt in the text box there.
14. If you have had difficulty with autofocus at high tilt, you can select the 'Focus every time above' option and fill in the desired angle above which you want to focus on every tilt.
15. If you have a centered image in buffer A, select the option to 'Align to image now in Buffer A'.
16. If you are starting at zero tilt, you should select the option to 'Refine eucentricity first' unless you have already done this.
17. If you are starting at high tilt and have acquired an anchor image on the way up, select the 'Use mid-tilt anchor' option and then use the up or down arrow if necessary to indicate which buffer contains the anchor (it should be in the right place if you used 'Walkup & Anchor').
18. Under 'Tracking control parameters', you typically need to consider only the first line, which controls whether Record images will be repeated if they are not centered well enough. If you never want this to happen, unselect the option. The percentage here will determine the width of the area near the edge of your reconstruction that is degraded because some images fail to contribute data to it. A value of 5% is probably reasonable for most uses. If the structures of interest nearly fill the camera frame at low tilt, then you might want to reduce this to 2.5-3%. If you are working at very high magnification, you might want to raise the value or unselect the option.
19. If you are working at high magnification where tracking will be relatively less precise, you may need to increase the value for 'Get tracking image when error in X/Y prediction is >' to avoid excessive tracking images. You may also need to increase the criterion for 'Get new track reference if Record alignment differs'.

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

How predictions work

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

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

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