Trial version

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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.

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

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

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

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.

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

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

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

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

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

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

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

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

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:

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

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

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

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

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

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

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

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

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

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

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

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.

It is possible to start a tilt series from a macro, which in turn makes it possible to run multiple tilt series automatically. In a future version, such series will be run directly from the Navigator, but for now the macro capability provides this functionality until all of the required features are worked out. In principle, a macro alone could be programmed to move the stage to each position and take a tilt series there, but in practice it will be more convenient to use the Navigator for moving to positions accurately. There are two ways that this can be set up:

1) If all tilt series have the same starting and ending angles and increment, then you should be able to set up a generic macro to run one tilt series and run that macro from the Acquire at Points command in the Navigator menu.

2) If it is necessary to have different angular ranges or increments for the series, then you will have to set up a macro that has commands for running each series, one after another.

In either case, each target position should be located within a medium-magnification Navigator map that is large enough to allow the position to be found reliably with the Realign to Item command. It is recommended that you also take a map at the magnification for the tilt series, and corresponding to a zero-degree view of the target area. Not only will this allow more precise positioning, it can also be used to guarantee that the beam and magnification settings are correct for starting a tilt series.

Cooker and Center are other macros for pre-exposing the area and recentering the beam (check the SerialEM download site for useful macros). It is advisable to set the working directory as shown, or include the full path to the directory in the command for opening the file. Before starting this acquisition, you would open the Tilt Series Setup dialog box via the �Setup Autostart� entry in the Tilt Series menu and set the angular range and any other needed parameters. The file opening command bases the file name on the label of the item in the Navigator list box, so you should also change the labels to generate suitable names. Make sure the target directory exists already and that none of the files already exist there. Mark the desired items as Acquire points. Finally, use the Acquire at Points command to open the Navigator Acquire dialog box and select appropriate options: select �Rough eucentricity� if the points are widely separated and maps are not already at approximately the right Z height; select �Realign to item�; �Restore scope state after align to item� can be turned off if there are high-magnification maps for each point; and of course select �Run macro� and dial up the right macro.

The rules for how to stop and restart operations with this arrangement are somewhat complex:

1) While the macro is executing a command other than the TiltSeries line, pushing �End� or �STOP� buttons on the Camera and Macro control panel will affect the execution of the macro and not the progress of the Navigator through the areas being acquired.

2) Once a macro is suspended with �STOP�, it can be resumed with the �Resume� button, or terminated with �End�, in which case the Navigator goes on to the next point.

3) Pushing �STOP� during the startup of the tilt series will terminate the tilt series and leave the macro in a suspended state on the TiltSeries line. If you then resume, you have the option of repeating the startup of the tilt series or skipping to the next line of the macro.

4) Pushing �STOP� or �End� after the tilt series has gotten past the startup will stop the tilt series as usual. You can then take any of the usual actions within the Tilt Series Controller, including resuming and terminating the series.

5) If you just terminate the series from the stopped state, the next line of the macro will be executed, which will lead to the Navigator going on to the next area. You will not have a chance to restart the series.

6) If you want to restart a tilt series after it has gotten past the startup, first STOP it. Then select �Stop� from the Macro menu, then terminate the tilt series. The macro will be suspended in a resumable state, and again you have the option of repeating the series or skipping it when you resume.

7) If you want the automatic acquisitions to stop after the current tilt series, select �End Acquire� in the Navigator menu.

Here, the first macro sets a variable with the item number, calls the second macro to do common tasks, then runs a tilt series. Note that items in the �ReportOtherItem� and �RealignToOtherItem� commands are referred to by their line number in the Navigator list box. If you are using this method, you may want to drag the target items to the top of the list box to avoid counting errors. The �restoreState� variable in the top macro should be set to 0 if the items being aligned to are maps with the magnification and beam conditions desired for the series; otherwise it should be 1 to restore microscope state after the realign operation. Set �roughEucen� to 0 or 1 depending on whether you need to run a rough eucentricity task at each area. If necessary, these variables could be set differently before calling the second macro for different series.

When running a tilt series just from a macro in this way, the rules for control are similar to above but a little simpler:

1) While the macro is executing a command other than the TiltSeries line, pushing the �STOP� button will suspend the macro.

2) Once a macro is suspended with �STOP�, it can be resumed with the �Resume� button, or just abandoned.

5) If you just terminate the series from the stopped state, the next line of the macro will be executed, which will lead to the macro going on to the next area. You will not have a chance to restart the series.

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

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

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

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

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

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

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

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

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

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

If the range of image shift required to make a montage is large, you can use stage movements instead. This is specified by an option in the Montage Setup dialog box. Montages constructed with stage movement will not fit together very well but are useful for getting an image of a large area.

The essence of low dose mode is that focusing and tracking operations for a tilt series are done in a location separate from the area being recorded, to spare that area from unnecessary beam exposure. In order to provide accurate focusing and tracking, these separate areas must be displaced along the tilt axis from the center of the recording area. SerialEM�s low dose mode provides five �areas� that can be defined to have different settings of magnification, spot size, and beam intensity, centered in up to 3 different locations. Four of them are linked to image acquisition and are named for the corresponding camera parameter set; the fifth provides a search mode that does not allow image acquisition with the main cameras. The areas are:

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

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

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

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

5. Search area � this area allows one other set of imaging parameters that is not linked to image acquisition. It could be used with a fast scanning camera or for scanning with the screen down at a lower magnification than is provided by the View area.

Although the Focus and Trial areas can be set up with completely independent parameters, it is most convenient to constrain them to be identical. This saves the effort of setting up the beam for both areas, avoids the image shift settling time required for getting between them, and minimizes the total range of image shift needed for low dose work. The latter factor is currently important on the 300 KV Tecnai because the objective aperture begins to occlude the image area for relatively small image shifts. Image shift is also quite limited on the JEOL 2100/2200 unless the scope has the high power image shift option.

The parameters that can be set separately for each low dose area are the magnification, spot size, beam brightness, and filter settings if there is an energy filter. The latter include whether the slit is in, and the slit width and energy loss. On the JEOL, the alpha setting can also be set separately. Absolute beam position is not stored, but a relative beam shift between areas can be set. Diffraction mode can be used for any area, although it is likely to be useful only for the Search area. If diffraction is used, the camera length is stored as a parameter, and on the Tecnai, the diffraction focus is also stored.

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

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

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

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

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

You can set the properties of a low dose area by making it be the current area then adjusting its features. The current area and its beam properties are displayed on the second line of the Low Dose control panel . There are two ways to set the current area: take an image of the area, or put the screen down and select the area with the �Area to show when screen down� radio buttons. For the search area, there is also a button to press, since there is no image acquisition in this area. You can adjust the properties of the current area by turning on the �Continuous update of mag & beam� checkbox. With this option on, any changes in the magnification, spot size, condenser lens setting, or energy filter settings will change the defined properties of the area. (Note that a relative beam position for the area is not updated; that is set with a separate control.) If the option is off, you can change these features temporarily, but the stored properties of the area will be reimposed next time a picture is taken of the area.

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

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

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

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

Your low dose parameters are saved to your settings file and will be restored when you restart SerialEM. If you have never started low dose before, the areas are undefined, and they will acquire the current microscope settings when they are first activated. Two sets of parameters are stored, one for a nonGIF camera and one for a GIF camera.

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

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

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

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

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

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

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

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

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

10) Make sure �Keep Focus and Trial identical� is selected and copy Record properties to the Trial area with the �T� copy button.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

The Navigator maintains a list of items which are shown in a box. It considers items to be of three types: points, polygons, and maps. A point is a single location, which will be displayed with a colored cross on images that contain the point. A polygon is set of connected points enclosing a region, which will be displayed as a set of lines forming a closed contour. A map is an image that has been saved to a file; the Navigator keeps track of where the image was saved as well as the stage position of the image and the conditions under which the image was acquired. The location of a map is displayed as a rectangle on other images. The Navigator makes it easy to reload a selected map image, regardless of whether all of your maps are in one file or separate files, and of whether the files are open or not. Maps can be either single frame or montaged images.

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

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

The large-area images used for maps are usually generated by taking montages with stage movements between the frames. The Navigator menu provides three ways to define the area that will be acquired in a montage. One option is simply to define an area that will capture the entire grid (�Setup Full Montage�). Another option is to move the stage to a series of points at the corners of the region of interest, such as the corners of the first and last sections of a ribbon. When each of these points is marked as a �corner� point, the �Setup Corner Montage� menu entry will set up a montage to encompass all of the area bounded by these points. The third option is to draw a polygon; the �Setup Polygon Montage� menu entry will set up a montage to capture the area delineated by the polygon. When you select one of these options, the Montage Setup dialog will open in a special mode in which it will adjust the number of frames to fit the selected area whenever you change the magnification or the overlap between adjacent montage frames. Note that in all of these cases, a full rectangular array of frames need not be acquired; the program will omit pieces that fall outside of the defined area.

The Navigator incorporates several features to help you get to a desired location despite the imperfections of the microscope. Image misalignments between the magnifications can become quite large at very low magnification. These misalignments limit the ability to mark a feature in an image at one magnification and go to that feature at a much higher magnification. If the image shifts needed to compensate for the misalignments have been calibrated, you can choose to have these image shifts applied so that features stay centered when you change magnification. Even if you do not turn on this option, the Navigator will use these image shifts to adjust stage positions appropriately for the particular magnification. With these adjustments, if you mark a feature in an image taken at, say 200x, and then go to that position while at a magnification like 3000x, you should be able to find the feature in an image taken at that magnification. In contrast, if you go to the position while still at 200x, the stage will be moved to the appropriate position for centering the feature at 200x, and it may not appear at the higher magnification. Note also that these adjustments are of limited accuracy and will probably not be adequate for keeping a feature in the field of view across a 100-fold range of magnification.

A second problem with returning to a marked position is that the actual stage position at which a feature is centered may change by a few microns over time, possibly due to drift, shrinkage, or accumulation of errors from extensive stage movements. There are two features that help deal with this problem. One is the ability to shift the stage positions of all of the items by the same amount. This can be done by adding a point at a defined feature in an existing map, then taking a current image that shows the feature and clicking on the feature there. The �Shift to Marker� menu entry is then used to shift the points. After this, you should be able to click on any point in that map and go to the point, without the error that was just eliminated by the shift. However, a different shift correction may be needed for distant points.

The second feature that helps in returning to a marked position is a routine for �realigning� to a position by correlation with an existing map taken in regular mag mode. In order to return reliably to a position, it must be located in a map that is at least as big as the maximum possible stage error, and preferably twice as large. Thus, points to be realigned to should probably be located in a map at least 10 microns in extent. They can be near the edge of such a map, since it is the overall size of the map and not the distance of the point from the edge that matters. Do not expect accurate realignment to a point in a map that is only 2 microns in extent.

If you wish to realign precisely to a high-magnification image, such as for doing a tilt series, then you will need two maps: the medium-mag map for reliably returning to the vicinity of the point, and a high-mag image stored as a map. There are two approaches to doing this. The simplest is just to take two map images at every desired location, the high-mag image and one at, say, 3000x. You can store these in the same file or different files. The other approach is to take advantage of an existing montaged map covering a larger area. In this case, the �Realign to Item� routine should be used before taking an image for the high-mag map, because of some subtleties in how errors in stage position are taken into account. Specifically, first add a point item at a desired position in the existing map, then go to the high magnification and run �Realign to Item�. Take an image, adjust the position as desired, and take a final image to store as a map. If you do not need to adjust positions after realigning, you can add multiple points, select �Acquire� for each one, then run the �Acquire at Points� menu item and specify �Realign to item� as an initial action..

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

To transform items to a new registration, you must first define some of the Navigator points at the first registration as registration points. You do this by selecting the �Registration point� checkbox and, if necessary, specifying a point number as well for each of the relevant points. (Registration point numbers will increment automatically.) After you have gone to a new registration, you then move the stage to the same positions, add a new Navigator point at each, and identify each point as a registration point with the same number as in the first registration. Once you have identified a set of corresponding points in this way, select a menu entry to transform the items from the first registration to the new one. The program will find a transformation whose complexity depends on the number of registration points available. Just a shift will be found with 1 point, a rotation and shift with 2 points, rotation, scaling, and shift with 3 or 4 points, or a full linear transformation including stretching with 5 points. All items except the registration points will then be transformed and associated with the new registration. (The registration points are left behind to allow the data to be transformed back if necessary.) If you just need to adjust by a shift after removing and replacing the holder or after moving the stage around a lot, you can use just one registration point, or use the �Shift to Marker� approach described above.

1. Go to a magnification that can be used to map the area of interest in a reasonable number of large pixels, on the order of 10K x 10K (e.g., 10 by 10 pieces, each 1K x 1K). This might be as low as 160x.

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

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

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

7. If necessary, shift the registration. Add a point on an identifiable feature on the low-mag map. Mark the same position on the new image with the left mouse button. Select �Shift to Marker�.

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

9. When you load an individual map after it has been acquired, you can simply click in the map then press �Go To Marker� to move the stage to the selected location. For more accurate positioning, go to the mag at which you want to view a feature, add a point item on the map, and use �Realign to Item�. You could then acquire a tilt series at high mag.

10. You do not need to close the montage file, but be sure to open a new file before trying to start a tilt series.

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

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

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

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

7. Select one of the points of interest in the Navigator list box and press �Go To XY�. You should be close to the desired point. Errors of 5-10 microns are likely if you localized the registration points in low mag mode; but the error should be much less if you localized them at higher mag.

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

Even when conversion to bytes is on, the acquisition of a new map will take 3 times as much memory as the number of pixels, because the scaling to bytes cannot be known until the whole montage is acquired. As soon as the montage is designated as a map, however, it will be converted to bytes and the extra memory will be available again. If the program cannot get memory for the initial overview image when acquiring a montage, it will increase the binning of this image to reduce the memory requirement. After you make such an image a map, you can then reload it to see it at full resolution, since this will require less memory.

If performance starts to suffer, use the Task Manager to assess the memory and swap file usage. Also notice the memory usage indicator in the Buffer Control Panel. To recover the memory occupied by a map in a higher buffer, make the image be the current one and use the Delete button in that control panel. If you are going to use the Navigator extensively, get as much memory as possible for the computer and make sure the swap file space is large (1 GB). Because the behavior of the computer is usually atrocious when significant swapping occurs, it is probably preferable to set the TotalMemoryLimitMB property to prevent the program from requesting large amounts of memory for montage overviews when memory use is near the limit (See General entries ).

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

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

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

MFC71.dll, msvcr71.dll - DLL�s for running under Windows 2000 (Tecnai version only)

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

• Set TietzCameraType to 2 for PXL camera (older F214, F224 with the PXL controller, 3 for PVCam camera (older Tem-Cam-F0124, F214 with the Model 300 controller from Photometrics), 4 for FastScan F114 camera using MVTitan PCI frame grabber, 5 for SCX camera (F224HD, single-port F415 with SCX controller), 6 for FastScan F114 camera using FireWire 1394 interface, 7 for ATC6 camera (very old F224), 8 for TemCam-F816, 9 for FastScan-F214 with Firewire controller, and 10 for TemCam-F415MP (4k camera with multi port readout)

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

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

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

You should start with a properties file that has a magnification table but does not have any �RotationAndPixel� lines in it. This step is almost unneeded on the Tecnai because the only variation that has been observed is in the rotation angles of the EFTEM table. The procedure is to start SerialEM and run the �List Mags� command from the �Calibration� menu. Compare this listing with the magnification table in the properties file. If it matches, you are ready to go on. If not, you need to cut and paste the output of �List Mags� into the properties file. On a Tecnai, step through the mags with the screen down and type in the mag that the Tecnai user interface shows. On a JEOL, the third field can be left blank or filled with 0�s.

On the Tecnai, now that you know the relation between mag index and mag, find out which is the lowest mag in M mode and verify that LowestMModeMagIndex is correct. On the JEOL, the routine will provide this value.

On the JEOL, following this magnification table will be a table of camera lengths for using diffraction mode. This table should be inserted in the properties file if it is not already there. The first line added to the properties file should be �CameraLengthTable nn�, where �nn� is the number of lines of camera lengths that follow.

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

The gain of an Eagle camera varies with binning, and it is necessary to calibrate the change in gain in order for dose calculations and intensity-related operations to work properly, including the acquisition of a gain reference. To do this, set the �Trial� parameters for a full-field dark-subtracted image at the maximum binning. Set the exposure time to 0.2 second and adjust the beam intensity so that �Trial� gives an image with counts between � and � of the saturation level. Take an image and use �Ctrl-I� to get the mean counts in the image. Repeat at each binning down to 1, without changing the exposure time.

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

If the camera controls only one shutter, skip the DigitalMicrograph script and just follow the procedure described in the next section. It is not so important but it will to provide a check on the timing for performing tasks after an exposure.

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

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

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

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

Measuring Camera Timing for Non-Gatan Camera or Gatan Camera with One Shutter (Priority 2)

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

The first Tietz camera that SerialEM was run with had a problem with gain-normalization of binned images, although F415 cameras have not shown this problem. The same problem has appeared on a 4K Eagle camera. Although the problem can be solved by making separate gain references for the different binnings, a more convenient solution (for the user) is to use �BinningOffset� entries in the camera properties section Here is a procedure that uses cross-correlation and does not require a trained eye to see the offsets:

1. Set camera parameters and the beam to take an dark-subtracted, unbinned 2Kx2K image with a medium number of counts. For a 4K camera, take a center half image. For a 1K camera, take a full image and divide the sizes below by 2.

3. Change to binning 2, reduce the exposure time appropriately, take an image, and save to bin2.st.

4. Repeat this procedure for each binning, taking the same area of the camera and saving to a separate file.

b. newstack �si 2048,2048 �ex M binM.st binMxM.st (for each higher binning M)

d. newstack �si 1024,1024 �ex M/2 binM.st binMxM/2.st (for each higher even binning M, where M/2 is a single digit, m divided by 2)

6. You can now correlate each fully expanded binned image against the unbinned image with:

7. Each correlation will end with a line like: �Maximum at ( -0.00, 0.99), transformation ( 0.00, -0.99)�. The binning offsets are the values of the maximum coordinate, rounded to integers. For example, if this result was obtained for binning 4, you would need an entry �BinningOffset 4 1 0 1� in the Properties file. The �4� refers to the binning of the image, the first �1� refers to the binning of the gain reference, and the next two numbers are the offsets in X and Y.

8. You can now correlate each higher even binning against the binned by 2 image:

9. Again, the offsets are the values of the maximum coordinate, but only if they are close to integer values. If they are, then you would add an entry like �BinningOffset 4 2 0 1�, where again the first number is the binning of the image and the second is the binning of the gain reference.

10. However, if an offset is close to halfway between two integers, then it is not possible to synthesize a good gain reference for the given binning from a binned by 2 reference. In this case, you need an entry like �BinningOffset 4 2 999 0�. The �999� tells the program not to make a reference for binning 4 from the binned by 2 reference.

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

This assessment should be done before the low magnification image shift and pixel size calibrations needed to use the Navigator. The ill-defined nature of focus in Low Mag mode means that it would be easy for a user to set a focus that creates two problems: 1) image scale might be significantly different from the scale at the focus at which calibrations were done; 2) the image position might depend on beam position to an extent that makes localization of positions problematic when using the Navigator. The solution is to use a standard focus for the calibrations and for mapping in Low Mag mode. This standard focus should be one that minimizes beam-position dependent image movements. The procedure is this:

1) With a specimen in place, go to a mag near the top of the Low Mag range and set the focus to Eucentric focus (on the Tecnai) or to the standard focus (on the JEOL).

2) Move the beam and watch how much the image moves. Change the focus until you see substantial image movement; vary the focus until you can tell whether the standard focus is near the point where image movement is minimized. If so, on the Tecnai, you are done; on the JEOL, return to standard focus and skip to step 4.

3) If image movement seems excessive at the standard/eucentric focus, adjust the focus to minimize the beam-dependent image movement.

4) Select Standard LM Focus in the Calibration menu to record this focus as the standard one for this magnification. It will be used at all low magnifications if it is the only such calibration.

5) If different focus levels are needed at different magnifications, repeat this procedure at multiple magnifications. Or, on the JEOL, go to the standard focus at the various magnifications and record the calibration.

The procedure described next involves calibrating the image shift at a series of magnifications and saving that information in the calibrations file, as well as taking a series of images and measuring the magnification and change in image rotation directly from them and entering this information into the properties file. How much of this you need to do depends largely on the type of microscope and on whether you intend to use the Navigator, as detailed in the table below. The goal here is to calibrate not just the image shift at each magnification but also the relationship between specimen and camera coordinates, specifically the pixel size on the specimen and orientation of the specimen axes on the camera. The basic principles underlying the calibration requirements are:

1) If image shift is calibrated at two magnifications and pixel size or axis rotation are known at one, the pixel size or rotation can be derived at the other, provided that the relationship between image shift and position on the specimen is the same at both magnifications.

2) Image shift calibrations are intrinsically somewhat more accurate and convenient than direct measurements of pixel size and axis rotation as described below.

3) It is thus preferable to calibrate image shift thoroughly and derive pixel size and rotation where appropriate, rather than the converse.

The minimal requirement is to measure one pixel size in each magnification range where the relationship between image shift and movement on the specimen is constant, and to measure the rotation of images between those magnification ranges. On the Tecnai there are just two such ranges, LM mode and regular mag mode. On the JEOL, there are often multiple ranges. In practice, it is recommended that redundant measurements be made.

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Tecnai: For work at regular magnifications, calibrate image shift from the lowest M-mode mag up to the highest mag where tilt series might be taken. It is recommended that you calibrate pixel size over the range of magnifications where users would typically expect an accurate pixel size, using the procedures described below. Relative rotations between mags need not be calibrated, but you should definitely enter a calibrated rotation angle for one mag at which tilt series are typically run.

To enable Navigator use, take an image of the calibration grating at the highest LM-mode mag and use this to measure the pixel size there and the rotation between LM and M-mode. Switch to a non-periodic specimen suitable for lower mag work and calibrate image shift at all LM mags down to the lowest mag that might be needed for grid mapping. Do the other steps listed in the table, described in other sections.

JEOL: Image shift shows discontinuities at a number of lower mags in the regular mag range and is not as constant as one might wish even within the ranges. Thus, it is necessary to calibrate image shift as well as measure pixel size and rotation between mags up to the highest mag at which tilt series might be run. A calibrated rotation angle is also needed for one mag at which tilt series are typically run.

To enable Navigator use, image shift calibrations as well as pixel sizes and relative rotations are needed throughout LM mode, down to the lowest mag needed for mapping. Before following the procedure below for regular mags, start at the lowest LM mode mag needed, go to standard focus, and take and save pictures of the calibration grating at each mag. You may be successful at calibrating image shift with this grating, but it is more reliable to use a specimen suitable for low mag work instead.

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

The procedure: This section describes the operations on the microscope for calibrating magnifications and image shifts.

1) Place a standard cross-line grating in the scope (2160 lines per mm). Use the kind with latex spheres, which are easier to focus and provide more protection against spurious cross-correlations.

5) Turn off �Align on Save� in the �Buffer� control panel (in the options section).

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

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

If you are going to do calibrations in Low Mag mode, first: (Tecnai): go to the highest LM-mode magnification and set the focus to eucentric focus. Take a Trial image and save it to the file. (JEOL): Go to the lowest LM mode mag needed and set the focus to standard focus. Adjust intensity, take a Trial image and save it to the file. Repeat at each mag up to the highest LM-mode mag.

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

4) Save the last image to the file, or take another Trial after adjusting intensity if desired. If you don�t want to measure pixel size and rotation at each mag, just save the image at the lowest M mode mag and at whatever other mags you want to measure pixel sizes at.

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

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

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

Before you start modeling anything, select Edit-Model-Header and change the entry in the �Pixel size� field to �1 pixels� if it is not already that. Otherwise you will not get contour lengths in pixels.

On each image, draw one contour with 4 lines (5 points), going along the grid lines and enclosing about as big a rectangle as you can. Make the last point coincide with the first. Count the number of squares in the two directions and insert the total distance around the perimeter, in squares, into the spreadsheet in the �Model Grid lines� column. For example, if you have 7 squares in one direction and 6 in the other, the total perimeter is 2 * (7 + 6) = 26. Use Edit-Contour-Info to find the length of the contour, or run imodinfo to get values for all contours after saving the model. Insert these values into the �Contour Length� column. You will see the pixel size in the �Unbinned cal pixel� column. The formula for the pixel size is (463 nm/square * number of squares) / (number of pixels * binning). Draw contours on the images until you reach a mag where you can no longer enclose 4 squares.

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

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

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

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

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

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

If you took a full set of images, copy the Midas rotation, magnification, and stretch values into the appropriate lines of the spreadsheet (the first image pair provides data for the second mag). The rotation is just there to keep a record of it, but the Midas mag and stretch are resolved into a single number, in the �Midas delta mag� column. You may find it easier to run:

This will list the rotation, magnification, and stretch values for each transformation in the file, as well as net magnification value on the right that you can put directly into the �Midas delta mag� column. For the lower mags, you can compare the �delta mag� values with the �Delta pixel� values to see how accurate these two methods are. The real use of the delta mag�s is to derive pixel sizes above the last mag where you could measure it directly.

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

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

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

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

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

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

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

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

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

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

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

Assessing Cycle Length of Stage Movement Errors: In the SerialEM Tools is a file named cycleMacro.txt containing a macro for assessing the error in stage movement over distances large enough to reveal the periodicity of these errors (this is described in more detail in Stage Shift Calibration ). You will need the specimen suitable for stage calibrations described there and in the next paragraph. Open a macro editing window with and Edit command in the Macro menu, and press the Load button to load this macro into the window. It will move the stage 132 microns in X and Y. Position the specimen in the middle of an area large enough in extent for this movement, and run the macro. The output in the log window will show the difference between the measured and requested movement as a percentage; the cumulative sum of these differences in microns, and the total sum of measured shifts in microns. If the error is more than a few percent, the periodicity should be apparent in either the percentage or the cumulative difference. If there is a repeatable pattern, determine the distance between the extreme points in the pattern over one or preferably two cycles, and divide by the number of cycles to get the cycle length. If these differ from the default values of 62 microns for X or 41 microns for Y, add lines for �StageCycleLengthX� and �StageCycleLengthY� to the property file. Do not bother if the percent error is only a few percent.

This step may be optional since the values reported by Pulokas et al. and also found on the Boulder Tecnais may be universal constants for the Compustage; and tests on one JEOL have shown no significant periodicity.

Low Magnification and Stage Calibrations: These calibrations are needed to do montaging with stage movement and to use the Navigator to acquire low and medium-mag maps. To calibrate stage shift, or image shift in Low Mag mode, you will need a specimen that is moderately rich in detail and that is either on a slot grid or on a grid with a very large mesh. Start at the lowest usable magnification in regular mag mode and find an area with good features. Run the Stage Shift command from the Calibration menu (read that entry for details). Drop to the highest mag in Low Mag mode. Repeat the stage calibration here, and also run an image shift calibration. The program will automatically the latter from scratch if it is the first calibration in LM-mode. The calibrations in LM-mode will be done at the eucentric focus, or at the standard focus setting on a JEOL if it has been entered into the property file. Continue going down in mag, doing the image shift calibration at each mag. Do 2 more stage calibrations, one at an intermediate mag and one near the lowest mag that might be used for mapping.

In LM-mode, at least on the Tecnai with slot grids and coarse mesh grids, strange image effects sometimes occur at the higher magnifications. These can be eliminated by inserting the smallest objective aperture that does not occlude the image on the camera.

Beam crossover calibration: This calibration should be done before the beam or spot intensity calibrations. Go to a high magnification like 100K, select Beam Crossover in the Calibration menu, and adjust the beam to crossover at each spot size.

Beam intensity calibration: This calibration is best done with no specimen. The procedure must be done for each spot size that will be used for tilt series; if low dose is going to be used, this might mean spots up to 6 or 8. In the latter case, try to calibrate up to whatever spot size gives 0.3 nanoamp of current. Start the procedure one or two steps above the highest mag that might be used (~200K for low spot size numbers, lower mags for the spots that will only be used for low dose?). An intensity calibration will work on only one side of crossover. You can calibrate one side of crossover based upon the users� most common practice. Alternatively, you can calibrate both sides of crossover provided that you have done the beam crossover calibration. See Beam Intensity for more details.

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

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

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

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

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

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

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

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

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

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

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

Transfer this stack and findhotpixels.com to a machine with IMOD 3.9 or higher. Edit findhotpixels.com to provide the name of the image stack, the name of a model file to place points in, a threshold for hot pixel strength, and a list of any hot columns that appear in the images, numbered from 0. Number the hot columns from zero. With a Gatan US 4000 camera you will need to exclude a large number of columns near the edges, 20 or more. Enter subm findhotpixels.

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

Add these lines to your properties file. If you entered any hot columns, you will also see one or more lines listing them as:

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

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

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

Once you have done that, you should do a separate calibration of image shifts and pixel sizes from the calibration on the upper camera (or at least do the image shifts and one pixel size.) SerialEM has some fancy logic for transferring calibrations from one camera to another, but I wouldn�t rely on it except for test purposes. Do a calibration of autofocus at one mag first, because then you will be able to use autofocus to focus when you switch between mags. Then follow the procedure above for getting the image shift calibrations and grating pictures.

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

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

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

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

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

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

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

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

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

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

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

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

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

Here is a procedure for setting up the program to be used extensively at a different voltage. It assumes you have a 300 kV scope and want to use it at 200 kV.

1. Make a subdirectory "200KV" in the SerialEM folder and copy SerialEMproperties.txt and SerialEMcalibrations.txt there - they will both need modification but they will provide a starting point.

2. Make a directory "200KV" or some other name in the area where your default SerialEMsettings.txt file is kept, i.e., the place where your standard shortcut for running SerialEM says to start the program.

4. Edit the new settings file and change the SystemPath from �C:\Program Files\SerialEM� to �C:\Program Files\SerialEM\200KV�

5. Make a copy of your shortcut for starting SerialEM, and rename it, e.g., �SerialEM-200KV�

6. Change the properties of this shortcut to "Start in" the directory where you placed your edited settings file.

7. Be sure to have an entry �SeparateGainReferenceKVs 200� in both the new property file and the standard one. You can also place �WarnIfKVnotAt 200� in the main property file and �WarnIfKVnotAt 300� in the 200 kV property file, to keep people from runnig with the wrong properties.

Now when you click on the new shortcut, the program will start with the new settings file and access the properties and calibrations in the 200KV folder. At this point, you can calibrate image shift, stage shift, autofocus, etc. You will also need to adjust the pixel sizes and rotations in the property file.

Each user who wants to use the program at that voltage would have to have the same kind of parallel arrangement of shortcuts and settings files. Either copy your special settings file or edit a copy of theirs to replace the �SystemPath�.

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

SerialEMProperties	The file must start with this entry.
GainReferencePath	The path to gain references, if it is not the system path.
MRCHeaderTitle	Title to be placed in MRC files; full title is SERIALEM: �your title� date-time
LogBookPathName	Full path name of a text file in which tilt series information will be logged. The �log book� contains tab-separated entries as follows: Date/time, image filename, starting Z value of tilt series, total number of Z values in the file, full image X size, Y size, file size in MB, starting angle, ending angle, tilt increment, COS for cosine increments or REG if not, minutes for tilt series, frames for montage or 1x1 for single-frame, camera name, nominal film mag, binning, pixel size in nm, LD for lowdose or ND if not, slit width if filtered or 0 if not, energy loss or 0 if not filtered.
StartupMessage	This entry can be added to provide a message upon program startup. The entry must be all one line but the message can consist of multiple lines, separated by \n. The message is printed to the log window if it is reopened, otherwise to a message box.
PrintStartupInfo	Set to 0 to stop program version, build time, and startup time information from being printed in the log window if it is open.
WarnIfKVnotAt	Use this entry with a KV value (e.g., 200) to get a warning message box if the program is started with the microscope at a different voltage.
ControlSetName # name	Use this entry to rename one of the camera control sets. Follow with the index (between 0 and 6; 0 to 4 are View to Preview) and the new name. The new name may not be substituted in all dialogs and message boxes and it may not all appear in a dialog if it is much longer than the name that it is replacing.
SearchAreaName	Use this entry to rename the Search area in Low Dose mode.
DisplayIsNot120DPI	Set this to 1 to get the right font size on a display that is not set for 120 DPI (large fonts).
ExitOnBadScopeStartup	Set this to 1 to have the program exit if errors occur when connecting to the microscope.
StartAsAdministrator	Set to 1 to turn on Administrator mode when starting the program.
MaxStackWindowSizeXY	This setting controls the maximum size of binned images that will be displayed in a separate window during a tilt series. Enter the maximum size for a square image; rectangular images will be binned to have no more pixels than a square image of this size. The default is 512.
TotalMemoryLimitMB	Limit on the total memory that the program should use for image buffers and camera reference images, in megabytes. Thus far, this limit is applied only when the program is requesting memory for a montage overview image, which can potentially be quite large if maps are being done. If the computer has 2 GB or memory or less, set this to the highest amount of memory that the program can compfortably use without causing swapping (e.g., perhaps 1200 for a 1.5 GB system). For a 3 GB system or a 4 GB system without the /3GB boot parameter, a value near 1900 might be appropriate to keep the program from running into the 2 GB memory limit. For a 4 GB system with the /3GB boot option set, this entry may not be needed, but 2700 would probably be a good value.
SkipGainRefWarning	Set to 1 to suppress the warning that the spot was normalized before starting to take a gain reference. This may be appropriate on a Tecnai F30.
FFTCircleRadii	Enter one or more values to customize the circles that can be drawn on a live FFT. The radius is expressed as fraction of the distance from the center to the edge of the FFT (Nyquist frequency). The default is one circle at 0.33.
SmallFontsBad	1 was needed to display small fonts properly on a laptop.
SingleTecnaiObject	0 to allow threads to open a second connection to the Tecnai; 1 to shut down the standard connection while a thread has a connection, and reopen it when done.
SimulationMode	Set to 1 to indicate a microscope simulator is being used (enables some actions on JEOL).
DebugOutput	Set to 1 to obtain general debugging output in the Log Window. Set to a list of key letters (e.g., JTC) to obtain additional output corresponding to the particular key letters. Key letters are: i for image shift-related items, l (lower case L) for low dose, L for more verbose low dose, u for update items when polling, e for event reports, and J for complete listing of JEOL calls, c for calibration and scale matrix derivation, b for beam shift, g for GIF, G for verbose GIF and magnification lookups (during JEOL updates), k for keystrokes, R for camera retraction, E for eagle camera, W for Winkler�s bug, and r for gain and dark references.
ExitWithUnsavedLog	Set to 1 to prevent being asked whether to save log before exiting.
TestGainFactors	Set to 1 to multiply images simulation by gain factors.

NumberOfCameras	This property has been discontinued.
ActiveCameraList	A list of all of the camera numbers for cameras that are active, in order as they are seen by DigitalMicrograph. The camera numbers can be from 0 to 3 and correspond to the numbers on the �CameraProperties� lines.
InitialCurrentCamera	The number of the camera that should initially be set as the current camera. Without this entry, the program will start with the first camera in the active camera list, or the first nonGIF camera if that is a GIF camera.
GainNormalizeInSerialEM	1 to perform gain normalization and dark subtraction in SerialEM or 0 to have all processing done in DigitalMicrograph or TIA. This is an initial, global setting that will be overridden by a setting for NormalizeInSerialEM in a specific camera�s properties. It can also be changed by the user on each instance of the program.
SeparateGainReferenceKVs	A list of KV values at which the program will automatically maintain a separate gain reference; e.g., �80 120� for a 200 KV scope. When the scope is close to one of these values, the program will attempt to access a gain reference file specific to that KV for gain normalization, and a new gain reference will be stored in a file specific to that KV. In addition, the program will not fall back to a DigitalMicrograph gain reference in the absence of a SerialEM reference. Do not include the standard operating KV of the scope in this list.
ReferenceMemoryLimitMB	The total memory allocated for dark references in SerialEM. Needs to be large for a 4K camera, e.g., 160 MB.
DarkRefAgeLimit	When gain normalization is done in SerialEM, a new dark reference will be taken if one is older than this time, in seconds.
GainRefInactivityLimit	When gain normalization is done in SerialEM, SerialEM will examine whether there is a new gain reference in DM if no image has been taken for longer than the given number in seconds. If DM gain references are being used, the new one will be fetched; if SerialEM gain references are being used, the user will be asked whether to use the newer one from DM instead. This number should be set a little less than the shortest reasonable time that it would for a user to switch to DM and take a gain reference.
DigitalMicrographReferencePath	The path to the folder where DM keeps gain references.
InnerXRayBorderDistance	When removing X-rays from a dark reference, the mean of surrounding pixels will be computed from pixels that are at least this distance from all pixels above the criterion.
OuterXRayBorderDistance	Pixels will be used to compute mean of surrounding pixels if they are no more than this distance from a pixel above the criterion.
XRayCriterionIterations	If a patch of points above the basic criterion is too big, then criterion will be raised for up to this many iterations to try to get the brightest subset.
XRayCriterionIncrease	On each such iteration, the criterion applied is the basic criterion times (1 + iteration number x this value).
GlobalExtraRotation	The angle to add to the rotation angle reported by the Tecnai to get the actual image rotation, i.e., the rotation of the tilt axis from the X axis.
RetractCameraOnEnteringEFTEM	1 to have an upper camera retract and the beam blanked during the retraction when EFTEM mode is turned on. This is necessary only if there is a pressure switch overriding the GIF�s blanking of the beam, because there is a delay between the time when the upper camera unblanks the beam and the time when the pressure switch allows the GIF to blank it again.
BlankWhenRetractingCamera	1 to have the beam blanked whenever any camera is retracted to unblock the path to a lower camera.
DefaultActAfterExposures	Set the starting or default value for the Post-actions setting that allows tilting and image shift to start during camera readout; i.e., 0 to disable post-exposure actions until the user enables them.
DefaultCameraDivide16BitBy2	Set to 1 to have all images from 16-bit cameras divided by 2 as they acquired in SerialEM. This is a default behavior that can be changed by each user.
MinimumBlankingTime	The time that it takes to blank and unblank the beam, measured by the Camera Timing Calibration routine. This entry is needed only if there is a Gatan camera with two shutters.
StartCameraInDebugMode	1 to turn on debug mode as soon as the connection to the camera is initialized. This will print all communication with the camera plugin to the DM Results window and also provide some verbose output to the SerialEM Log Window.
TietzFilmShutterSwitchTime	Seconds to wait after switching the Tietz camera to film shutter mode; this time specifies the minimum pre-exposure time that will occur on the first acquisition after switching to film shutter.

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

CameraProperties	This entry, followed by a camera number from 0 to 3, signals the start of a section of properties for a camera, which can include any of the entries in this table.
Name	The name of the camera, which will appear in the camera, montaging, and tilt series setup dialogs. Spaces are allowed.
TietzType	0 for a non-Tietz camera, 1 for Tietz simulation camera, 2 for PXL camera (older F214, F224 with the PXL controller), 3 for PVCam camera (older Tem-Cam-F0124, F214 with the Model 300 controller from Photometrics, also some 4k F415MP multi-port readout), 4 for FastScan F114 camera using MVTitan PCI frame grabber, 5 for SCX camera (F224HD, single-port F415 with SCX controller), 6 for FastScan F114 camera using FireWire 1394 interface, 7 for ATC6 camera (very old F224), 8 for TemCam-F816, 9 for TemCam-F415MP (4k camera with multi port readout), and 10 for FastScan-F214 with Firewire controller.
AMTcameraType	1 for an AMT camera. AMT cameras cannot coexist with Gatan cameras.
FEIcameraType	1 for an FEI Eagle camera. This entry implies RestrictedSizeType 1. It also implies a MinimumExposure of 0.011, unless a different value is entered.
RestrictedSizeType	1 if acquisitions are restricted to full, half and quarter centered images.
TietzCanPreExpose	Set this entry to 0 or 1 to indicate whether a Tietz camera has a shuttering mode that allows pre-exposure of the specimen, using the film shutter and beam blanker. This setting works only if the EMShutter module is installed and an appropriate Tietz shutterbox is being used.
TietzGainIndex	Gain index to use for a Tietz camera. This is not the gain itself but a number between 1 and the number of gains available. The default is 1.
DMGainReferenceName	The full name of this camera�s DM gain reference, including spaces and .dm3.
CameraSizeX	Number of pixels in X
CameraSizeY	Number of pixels in Y
XMustBeMultipleOf	This entry constrains the size in X of images to be acquired to be a multiple of the given number. This entry is overridden by a RestrictedSizeType entry.
YMustBeMultipleOf	This entry constrains the size in Y of images to be acquired to be a multiple of the given number. This entry is overridden by a RestrictedSizeType entry.
CoordsMustBeMultiple	Set this entry to 1 if the individual coordinates (left and right in X, top and bottom in Y) must also be a multiple of the values specified in XmustBeMultipleOf and YmustBeMultipleOf, respectively.
FourPortReadout	Set this entry to 1 for a 4-port readout camera.
BasicCorrections	This entry determines the corrections done for all image acquisitions in DigitalMicrograph. It can be the sum of 1 for defect correction, 16 for bias correction, and 32 for linearization. The default value if none is entered is -1, which means that default corrections will be done for the particular camera.
MakesUnsignedImages	Set this entry to 1 for a 16-bit camera that produces unsigned 16-bit integer images.
CountsPerElectron	The camera gain, i.e. the number of counts per primary electron. This entry is needed for dose estimates. It should be in counts before dividing by 2 for a 16-bit camera.
Binnings	A list of valid binnings for this camera. Up to 9 binnings may be listed and will appear in the camera setup dialog.
BinningOffset	This entry may be needed to make gain normalization work well in binned images when gain normalization is being done in SerialEM. Follow the keyword with 4 numbers: the binning of images being normalized, the binning of the gain reference (1 or 2), the offset in X (a small positive or negative number), and the offset in Y. Offsets may be needed for Tietz and Eagle cameras. The appropriate offsets will be set up automatically for 4-port readout cameras for binnings that are not a power of 2, but these entries could be used to override the assumed settings. To prevent the binned by 2 reference from being used with a higher even binning, enter a line with �binning 2 999 999�.
RelativeGainFactors	A list of relative camera gain values, one per binning listed on the Binnings line. This entry is needed for the Eagle camera. The factor should be 1 for the binning at which counts per electron is calibrated.
ExtraGainReferences	Enter the number of binnings above 2 for which to allow separate gain references to be acquired. Up to 4 additional gain references may be saved, for the four binnings following 1 and 2 on the �Binnings� line. These references may be useful if there are problems with quadrant correction on four-port cameras and if the imbalance between quadrants differs between binnings.
NormalizeInSerialEM	Initial setting (0 or 1) for whether images should be gain-normalized in SerialEM. A value for a specific camera overrides the global default value provided by GainNormalizeInSeriaEM.
UsableArea	Specifies the coordinates of the usable area on the CCD, in Digital Micrograph coordinates numbered from 0. Follow the keyword with the top, left, bottom, and right coordinates of the last good column or row on the chip. This is needed if gain normalization is being done in SerialEM.
SubareasAreBad	Set this entry to 1 for a Tietz camera if taking an image of a subarea makes the next image of the full camera area, or a much larger subarea, be bad.
BadColumns	Specify a list of bad columns that will be replaced in all processed images (gain normalized or dark subtracted). List the columns in increasing order using DM coordinates numbered from 0. This is needed if gain normalization is being done in SerialEM.
PartialBadColumn	Specify one or more adjacent columns needing correction over a limited extent in Y. Enter four numbers: the starting column number (numbered from 0), the number of columns, the top Y coordinate (numbered from 0 at the top of the image), and the bottom Y coordinate. Use multiple lines to enter multiple corrections.
HotPixels	Specify a list of hot pixels if X ray removal from dark references is going to be used, to prevent these pixels from being treated as X rays. Each pixel is specified by an X and a Y coordinate. Multiple pixels can be entered on one line, and multiple lines can be entered.
HotPixelsAreImodCoords	Enter 0 or 1 if hot pixel coordinates are numbered from 0 (DM coordinates) or from 1 (IMOD/3dmod coordinates) respectively.
HotColumns	Specify a list of hot columns which should not be analyzed for X rays, in DM coordinates numbered from 0. Include the bad columns if they are hot.
DarkXRayAbsoluteCriterion	The absolute number of counts by which a pixel must exceed the mean of a dark reference to be treated as an X ray. These should be counts before division by 2, if any, for a 16-bit camera.
DarkXRaySDCriterion	The number of standard deviations above the mean that a pixel value must be to be treated as a dark reference X ray.
DarkXRayRequireBothCriteria	Enter 1 if a pixel must exceed both the absolute and the standard deviation criterion to be treated as a dark reference X ray, or 0 if exceeding either criterion will do.
MaximumXRayDiameter	The maximum diameter of an area that will be replaced as an X ray in a dark reference, in unbinned pixels.
ShowRemoveXRaysBox	Set to 1 to enable the appearance of the �Remove X-rays� checkbox in the camera setup dialog. This option should be enabled only if the particular camera generates images where X-rays can ruin the tracking.
BeamBlankShutter	The shutter number of the beam blanking shutter (0 or 1; a Tecnai should be set up so that this is 1.)
OnlyOneShutter	Set this entry to 1 if there is only one shutter driven by this camera.
SetAlternateShutterNormallyClosed	1 to issue a command upon initialization to make sure that this camera is configured so that the beam shutter is normally closed and the film shutter is normally open.
BuiltInSettling	The intrinsic pre-exposure time in seconds when using the regular shutter for a Gatan camera; this seems to correspond to the clear time of the camera.
StartupDelay	Average time in seconds between issuing a command to take a picture, and the start of the actual exposure sequence (clearing the CCD for a Gatan camera, or starting the exposure for a Tietz camera).
MinimumDriftSettling	The smallest drift settling that SerialEM should try to produce. For a Gatan camera. it corresponds to half of the range of variability in the time that an exposure starts after image acquisition is requested by SerialEM. [For a Tietz camera, this should be set to 0 or a small number like 0.01].
ExtraBeamTime	The amount of time to allow after the expected end of the exposure before microscope actions can be taken and the beam can be blanked the beam to avoid beam exposure during the readout. For a Gatan camera, this should equal MinimumDriftSettling. For a Tietz camera, the sum of StartupDelay and this timing factor is used to control how soon microscope actions can be taken after readout should begin, but no beam blanking is needed.
ExtraOpenShutterTime	When the shutter is opened to provide a pre-exposure that exceeds the built-in settling (pre-exposure), the time specified by this entry is subtracted from the additional pre-exposure desired to keep the pre-exposure from exceeding the desired length.
ShutterDeadTime	The amount of exposure time lost when the beam shutter is operated; an entry for this is needed on JEOLs and can be obtained by running the routine in the Calibration menu.
MinimumExposure	Minimum exposure time that the camera will allow.
Retractable	Set to 1 to indicate that the camera is retractable
InsertionDelay	Delay in seconds after inserting the camera.
RetractionDelay	Delay in seconds after retracting the camera.
CheckStableTemperature	Set to 1 to check for whether the camera is at a stable temperature before trying to acquire. Once the camera passes the test, the program will not check again. (For Gatan camera of controller class �PICM�, such as Orius).
SideMounted	Set to 1 for a side-mounted camera above the viewing screen, so that the program will not raise the screen before taking a picture.
GIF	Set to 1 to indicate that this is a GIF camera
Order	An arbitrary number to indicate the order of the camera in the beam path; cameras with lower order can block the beam from reaching cameras with higher order.
HasTVCamera	Set to 1 to indicate that this GIF has a TV camera.
InsertTVToUnblank	Set to 1 to indicate that the TV camera must be inserted to keep this camera from blanking an upper camera. This setting is needed with a GIF unless you have some other means to keep the GIF from blanking when the upper camera is in use.
FilmToCameraMagnification	Magnification ratio between the film plane and the camera. This is used to provide a fallback pixel size if pixel size is neither directly calibrated nor deducible from image shift calibrations.
PixelSizeInMicrons	Actual size of unbinned pixels in the CCD chip.
ExtraRotation	Number that needs to be added to the Tecnai-reported rotation angle and the global extra rotation angle too determine image rotations for this camera. Only relevant if rotations are not calibrated directly or indirectly.
RotationAndPixel	This entry specifies a set of calibration values for a single magnification. It should contain the following numbers on the line: 1) Magnification index 2) Change in image rotation from the next lower mag to this mag (in degrees, CCW positive), or 999 if this was not measured 3) The measured rotation from the X axis to the tilt axis (in degrees, CCW positive), i.e., the rotation angle solved for in Tiltalign plus 90 degrees; or 999 if this was not measured. 4) The pixel size in nanometers, or 0 if this was not measured.
EndCameraProperties	This entry indicates the end of the block of properties for a camera.

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

DeltaZtoDefocusFactor	Factor needed to adjust for a discrepancy between the change in defocus reported by the microscope and the actual defocus change. Determined by calibrating autofocus, moving in Z and measuring defocus; the factor is (defocus change)/(change in Z height)
StandardLowMagFocus	A standard focus value for doing montages and calibrations in LM mode. On the Tecnai, a value of 0, representing �eucentric focus�, should be adequate, unless a different focus minimizes image movement when the beam is moved. With a value of -999, no standard focus is asserted when montaging or calibrating. The value for a particular focus setting can be determined with the ReportFocus macro command. The default is 0 for a Tecnai and -999 for a JEOL. This entry is overridden by any calibrations made with Standard LM Focus in the Calibrate menu.
ScreenCurrentFactor	Factor needed to give a calibrated current in the screen meter
SmallScreenFactor	Additional factor needed to get the same current reading when switching between main screen and small current, with the beam filling the main screen
LensNormalizationDelay	Time delay after projector lens normalization, in milliseconds
LowestMModeMagIndex	Index of the lowest magnification in M mode, needed to prevent the task routines or filter control from switching into LM mode, and to indicate that image shift calibrations are not congruent across the LM-M boundary
LowestGIFModeMagIndex	Index of lowest magnification that should be used for tasks with the GIF camera. The default is to use the LowestMModeMagIndex.
NumberOfSpotSizes	Number of spot sizes on the microscope; default is 11.
IntensityToC2Factor	This entry is no longer needed; instead, the factor is measured by a calibration procedure and stored in the Calibrations file
TiltAxisOffset	Distance from the optical axis to the tilt axis, as indicated by the Refine Eucentricity routine. To determine this value, run the routine 3-5 times near (within 100 microns) of the center of the grid.
StageLimits	Limiting coordinates for stage movement in microns: minimum and maximum X coordinate, and minimum and maximum Y coordinate. The default is +990 or -990 microns; a 0 may be entered to use the default.
StageMoveDelay	Delay time in milliseconds from moving the stage to taking a picture.
InvertStageXAxis	Set to 1 to indicate that the stage coordinate system is left-handed when projected onto the screen. The axes of this coordinate system are assessed by noting the direction that the image moves when stage coordinate is increased in X or Y; if the direction for X is 90 degrees counterclockwise from the direction for Y then this property is needed (and a global rotation of 180 degrees may also be needed.)
RotateHeaderAngleBy180	If the tilt axis rotation angle defined by these properties results in reconstructions with inverted handedness, set this entry to 1 to rotate the angle output to the image file header by 180 degrees and restore the handedness. This will probably be needed if InvertStageXAxis is set.
FloatingCurrentMeterSmoothed	0 for no smoothing or 1 for smoothing of current readings when the floating current meter is opened
CurrentMeterLogBase	Base value added to screen current in nanoamps, before taking the log for smoothing. Adjust this to make the smoothing behave similarly for large and small currents.
CurrentMeterSmootherThreshold1	Threshold change for a single value to trigger a change in the displayed, smoothed value (in log units).
CurrentMeterSmootherThreshold2	Threshold change in the running mean that will trigger a change in the displayed value (in log units). Change these two thresholds together to adjust smoothing.
WatchGauge	Add a gauge to be watched for the VAC indicator in the microscope property panel. Follow this entry with the official name of the gauge (e.g., column vacuum IGP1 is P4), the threshold between a green and yellow reading, and the threshold between a yellow and red reading, in Pascals. A formula based on several simultaneous observations of Pascals and FEI log units for IGP is: Log reading = 26.2 * log(Pascal reading) + 135 Or, Pascals = 10 ** ((log reading � 135) /26.2)
MicronsPerUnitImageShift	A parameter that is used in the initial calibration of image shift (or when calibrating �from scratch�). If image shift is not properly calibrated on the scope, this may need to be set to something besides 1.
ImageShiftLimit	Limit on image shift at regular magnifications; image shifts are tested against this limit in some but not all situations. The default is 15 microns.
LowMagShiftLimit	Limit on image shift in LM mode; image shifts are tested against this limit in some but not all situations. The default is 150 microns.
MaxCalibrationImageShift	Maximum image shift change when calibrating image shift
MaxLMCalibrationImageShift	Maximum image shift change when calibrating image shift at LM-mode mags.
ISoffsetCalStageLimit	Maximum stage shift allowed during calibration of image shift offsets between magnifications. The default is 0.025 micron.
StageCalibrationBacklash	Microns of movement to adjust for backlash in the stage calibration procedure. The default is 3 microns but 10 microns was needed on a JEOL.
StageCycleLengthX	Cycle length at which modulations in position error repeat for stage movements in X, which is used to control the extent of stage calibrations in X. Calibrations will traverse a multiple of this distance in X unless limited by the MaxStageCalExtent or by the possible number of steps. The default is 61 microns (found on two Tecnais).
StageCycleLengthY	Cycle length of modulations in stage position error in Y, used to control the extent of stage calibrations in Y. The default is 42 microns.
MaxStageCalExtent	Maximum distance over which to do the stage calibration. If this is larger than the cycle lengths in X and Y, it will allow calibrations to go over one cycle length, but may limit the number of cycles traversed at very low mag. A value lower than a cycle length will keep the calibration from being done over a full cycle on that axis. The default is 140 microns, which limits the extent to 2 cycles in X and 3 cycles in Y with the default cycle lengths.
UseTrialSizeForShiftCal	Set to 1 to use the image size in the Trial control set for image and stage shift calibrations.
MagnificationTable	A table of magnification index, film and screen magnifications, and the rotation angles reported by the Tecnai. The line with this entry must contain the number of table entries to follow, then there must be that many lines following. The form of each line is: Index Film mag Rotation angle Screen mag The first three entries can be obtained by selecting List Mags in the Calibration menu. The screen mag must be added by hand. On a JEOL, the third entry can be omitted unless there is a GIF, in which case a 0 should be entered. If there is a GIF, each line should have three more entries: Mag with screen up Rotation angle Mag with screen down Again, the third entry can be omitted on a JEOL.
CameraLengthTable	A table of camera lengths, needed on the JEOL to recognize diffraction mode. The line with this entry must contain the number of table entries to follow, then there must be that many lines following. The form of each line is: Index Camera length in mm This table is obtained by selecting List Mags in the Calibration menu and appears after the magnification table.
ImageShiftDelays	A table of delays to apply after an image shift. The delays are applied before taking an image, and they are scaled up or down in some situations. They are also adjusted up or down for camera startup and clearing times lower or higher than 0.8 seconds. The line with this entry must contain the number of entries to follow, then there must be that many lines of the following form: Distance in microns Delay in seconds
ExtraISdelayPerMagDoubling	This entry controls an increase in the image shift delay at magnifications where the pixel size of a Record image is smaller than 1 nm. With the default value of 0.25 second, 0.7 * 0.25 is added to the delay with a pixel size of 0.75 nm, 0.25 is added for 0.5 nm, 1.4 * 0.25 for 0.375 nm, 0.5 is added for 0.25 nm, etc.
LowDoseBeamNormDelay	In low dose mode, the condenser lenses can be normalized by passing through the intensity and spot size of the View area when changing areas (needed on some JEOL scopes). This value sets the delay in milliseconds after going to the View parameters (default 100 ms).

CheckAutofocusChange	The change in defocus that will be applied in the positive and negative direction when the routine to check autofocus is run.
ResetRealignMaxIterations	Maximum number of iterations when the routine to reset image shift and realign is run.
ResetRealignIterationCriterion	If image shift exceeds this criterion after resetting the image shift and realigning, another iteration will be done, up to the maximum number of iterations.
ResetRealignHigherMagCriterion	If image shift is less than this criterion when doing the reset and realign task, then the alignment pictures will be taken at one mag step higher than the default.
WalkUpMaxInterval	Maximum tilt interval for the walk up task.
WalkUpMinInterval	Minimum tilt interval for the walk up task. Intervals will initially be the maximum interval times the cosine of the tilt angle, but will not become smaller than this minimum.
WalkUpShiftLimit	When image shift exceeds this limit during the walk up task, the reset and realign operation will be run.
WalkUpLowDoseISLimit	A similar limit on image shift, applied when walking up in low dose mode.
EucentricityBacklashZ	Amount to move in Z to correct for backlash when changing Z during the eucentricity routines.
EucentricityCoarseInitialAngle	Starting tilt angle for the coarse eucentricity task.
EucentricityCoarseInitialIncrement	Starting tilt increment for the coarse eucentricity task.
EucentricityResetISThreshold	Image shift will be reset to zero, and the stage moved to compensate, if it exceeds this criterion before a eucentricity task.
EucentricityCoarseMaxTilt	Maximum tilt angle for the coarse eucentricity task.
EucentricityCoarseMaxIncrement	Maximum tilt increment for the coarse eucentricity task.
EucentricityCoarseTargetShift	The coarse eucentricity routine will increase the tilt increment to try to make the image shift by this amount in microns.
EucentricityCoarseMaxIncrementChange	Maximum factor by which the tilt increment will change from one trial to the next in the coarse eucentricity task.
EucentricityFineIterationLimit	Maximum number of iterations for the fine eucentricity task.
EucentricityMaxFineIS	Maximum image shift for the fine eucentricity task before it changes Z height and starts a new iteration.
ResetRealignMinField	Minimum camera field of view, in microns, for resetting image shift and realigning, which determines the mag used.
ReverseTiltMinField	Minimum camera field of view, in microns, for the reverse tilt task.
EucentricityCoarseMinField	Minimum camera field of view, in microns, for the coarse eucentricity task.
EucentricityFineMinField	Minimum camera field of view, in microns, for the fine eucentricity task.
EucentricityFineAlignMinField	Minimum camera field of view, in microns, for doing fine eucentricity and maintaining image alignment.
WalkUpMinField	Minimum camera field of view, in microns, for the walk up task.
TiltAfterMoveMinField	Minimum camera field of view, in microns, for realigning after a tilt following a stage move. If the value is non-zero, this task is invoked during walk up and tilt series on the tilt after an image shift reset. The default is 0. for Tecnai and 4.5 um for JEOL.
TiltBacklash	Amount to tilt to eliminate tilt backlash.
MinTaskExposure	Minimum exposure time allowed, when it is necessary to reduce the exposure time upon going to a lower magnification (i.e., when intensity cannot be reduced enough). This minimum will apply both for tasks and for low magnification tracking in a tilt series. The default is 0.002, but a higher value may be needed if camera shuttering is slow and exposures of this duration give no image
RefineZLPStepSize	The step size in eV for the Refine ZLP procedure. Default is 2.
RefineZLPSlitWidth	The slit width used for the Refine ZLP procedure. Default is 20 eV.
RefineZLPMinimumExposure	The minimum exposure used in the refine ZLP procedure, in seconds. The procedure uses the Trial parameter set in normal mode and the Preview parameter set in Low Dose mode, with the exposure reduced by a factor of 10, but not below this minimum. The default is 0.005, but numbers as low as 0.002 can be used if the number of counts is consistent within a few percent from one exposure to the next.
RefineZLPMaxMeanRatio	The maximum ratio between the final and peak mean counts in the refine ZLP procedure, i.e. the counts after the slit is moved to the left of the ZLP must fall below the peak counts when the slit is over the ZLP by this ratio or less. Raise this ratio if the ZLP fails to be recognized. The default is 0.02.
EnergyShiftCalMinField	Minimum camera field size for the lowest mag that will be done when calibrating mag-dependent energy shifts. This should be set to the largest minimum field size for any of the tasks. The default is 8 microns.
RealignItemMinMarginNeeded	When running Realign to Item, Navigator must find a map containing the item with at least this much distance from the center of an image frame to the edge of the map. The default is 1.5 microns.
RealignItemMaxMarginNeeded	When running Realign to Item, maps with a greater distance from the center of an image frame to the edge of the map will be preferred for the first round of aligning to a frame of the map; but all distances greater than the value here will be treated equally. The default is 10 microns.
RealignItemMinMarginWanted	When running Realign to Item, a frame that is closer to the target will be preferred for the first round of alignment as long as the distance from the frame center to map edge is bigger than this value. The default is 5 microns.

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

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

JeolUpdateByEvent	Set to 0 to disable updating by events if this screws up.
UpdateSpectroscopyByEvent	Set to 1 if the JEOL interface makes the promised event call when changing between spectroscopy and imaging modes.
InitializeJEOLDelay	The delay between initializing the JEOL COM object and the first attempt to call functions, in milliseconds, if events are not being used for update. Without this delay, errors occur.
ScopeUpdateInterval	The interval in milliseconds between calls to the function that reads scope properties and updates the user interface. The default is 100. In the JEOL version, the update function does not normally access the microscope so this should be OK.
JeolUpdateSleep	The sleep time, in milliseconds, between calls to the update function that actually queries the scope in a separate thread. If events are used for update, this function does one or two queries per call, taking up to 150 msec, so this interval must be long enough to leave substantial time for user interface handling, etc. If events are not used for update, the stage position and status calls take up to 300 msec.
JEOLReportsSmallScreen	Set to 0 to indicate that the scope does not report the small screen position. Needed on 2100.
JEOLReportsLargeScreen	Set to 0 to indicate that the scope does not report any detector at all representing the large screen position. Do not set this to 0 if an alternative main detector ID can be set.
JeolMainDetectorID	Set this to indicate the ID of a detector whose position is reported and which needs to be retracted for camera imaging. The default ID is 13 for the main screen. Possible alternative IDs are 5 for upper TV camera and 17 for a lower TV camera.
JeolControlsBeamValve	Set to 0 to indicate that the gun valve cannot be controlled (non-FEG).
HasOmegaFilter	Set to 1 to indicate that the scope has an omega filter. The program will then enable the filter control panel but not do any of the mag changes, etc, involved in EFTEM mode.
JeolHasNoAlpha	Set to 1 to indicate that this microscope has no alpha setting. This seems not to be necessary but could be set if errors are generated from attempts to read or set alpha. This setting is implied when Jeol1230 is set.
Jeol1230	Set to 1 for a JEOL 1230; has many implications.
FocusTickTable	Use this entry to start a table of the number of focus ticks for one click of the focus knob at various magnifications. Follow with the number of lines to follow. Then on each following line, put a mag index and the number of ticks. The default is 1 for any mag not entered here.
JeolForceMDSmode	Set to -1 to turn off MDS mode and give a warning message upon program startup, or to 1 to force the microscope into MDS mode so that image shift will not be reset when magnification is changed (1 is obsolete).
JeolPostMagChangeDelay	Some versions of TEMCON generate errors when stage parameters are accessed too soon after a magnification change. This entry can take one or two delay times in milliseconds. The first number specifies the delay for any scope accesses after the program changes mag. The second number provides for an additional time during which access to the stage will be deferred. If only one number is used, values of 2000 or more may be needed to prevent errors. With two numbers, values of 1000 and 3000 could be tried. Default is 0.
MagChangeFixISdelay	If updating by event, the maximum possible interval from the time when a neutral image shift is reported to the time when the mag change responsible for the neutralization is reported.
OtherShiftBoundaries	List of mag indexes at which image or beam shift calibrations are not congruent with the next lower mag. Just like LowestMModeMagIndex, such an index indicates the start of a new mag range. Within a mag range, both the relation between image shift output and position on the specimen, and the relation between beam shift output and position on the specimen, should be constant.
JEOLStageMotorRounding	The value to which stage coordinates will be rounded before being used to drive the stage motors, in microns. Should be 0.001; with the emulator 0.1 is needed.
JEOLStagePiezoRounding	If this value is non-zero, then after stage coordinates are rounded as indicated by JEOLStageMotorRounding, the remainder will be rounded by this value (in microns) and used to drive the piezoelectric positioners. With a value of zero, no piezo positioning will be done. Keep this at 0.
JEOLObjectiveLensToMicrons	Scale factor for converting the objective lens fine digital readout to microns of defocus. The default is 0.0058, the value for 300 KV; it may need to be changed to make the defocus values in SerialEM match the values on the microscope display. For 200 KV the value should be 0.002.
JEOLObjectiveMiniToMicrons	Scale factor for converting objective minilens values to microns in low mag mode. The default is 0.01 (measured on a 2100).
JEOLBeamShiftToMicrons	Scale factor for rough conversion of beam shift values to microns. The default is 0.025, the value for 300 KV, which also works at 200 KV. It needs to be changed to get big enough spot movements in the first round of calibrating beam shift.
JEOLLowMagBeamShiftToMicrons	Scale factor needed for calibrating beam shift in low mag mode. If this is omitted, the factor specified in JEOLBeamShiftToMicrons is used. A value of 0.15 works at 200 KV.
MicronsPerUnitImageShift	A parameter that is used in the initial calibration of image shift (or when calibrating �from scratch�). The right value is needed to get shifts that are big enough, but not too big, in the first round of calibration. A value of 0.02 is needed at 200 KV; a value of 0.1 is needed at 300 KV.
LowMagMicronsPerUnitIS	Scaling factor needed for initial calibration image shift in low mag mode. If this omitted, the factor specified in MicronsPerUnitImageShift is used. A value of 1 is needed at 200 KV, 1.5 at 300 KV.
JeolUseProjectorForIS	Set to 1 to substitute projector shift for image shift. Neutral value, image shift, and beam shift calibrations would need to be redone and kept in a separate calibration file when operating in this mode, and different values of the microns per unit image shift values may be needed.
JeolMMperUnitProjector	When using projector shifts, this factor is used in the initial calibration of image shift; it specifies the approximate number of millimeters of shift on the camera per initially scaled unit of projector shift. The default is 1.5, based on scale matrices on a 2100 in which one unit of projector shift produced ~100 15-micron pixels of shift.
MessageBoxWhenClipIS	Set to 0 to avoid having a message box appear when image shift goes beyond range and gets clipped to its limiting values.

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

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

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

You can use this command to open a new image file even if another file is already open. The newly opened file becomes the current file for reading and writing. The current file can be selected with a spin button in the Buffer Control Panel. The name of the current file is always displayed in the program�s title bar

You can open an existing image file for reading and writing with the Open Old command. You can also read from a file without opening it, using the Read from Other command.

Use this command to open an existing image file for reading images from and saving images to. You will enter the File Open dialog box to select a file. If the file is a montage, then montaging will be activated. You can use this command to open a file even when another file is open. The file that you open becomes the current file for reading and writing.

Use this command to open a new MRC file for saving montages. You first enter the Montage Setup dialog box, which allows you to specify the number of frames, magnification, and binning. From there, you enter the File Properties dialog box then the Save As dialog box. A new montage can be started even when there is already an open file; the new file becomes the current open file and montaging is activated with the parameters for this file.

Use this command to open a file for montaging, or to check parameters if the current open file is a montage. In either case, you first enter the Montage Setup dialog box. If you are starting a new file, you then enter the File Properties dialog box then the Save As dialog box.

Use this command to close the current open image file. If there is more than one file open, the file with the next lower number becomes the new current file. Montaging may be turned on, or terminated, depending on the type of that file.

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

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

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

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

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

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

Use this command to modify the truncation of the data when saving images as bytes. When one chooses to save bytes, then SerialEM can avoid compressing the dynamic range of the data by truncating the values of a relatively few pixels. The number of pixels to truncate at the black and white end of the range is initially specified in the File Properties dialog, but can be changed thereafter with this command. Using this command sets the values for the current open file and the default for newly opened files but does not affect any other open files.

Use this command to modify the way that data from a 16-bit camera will be treated when they are saved as integers in a signed data file. You will be asked to enter 0 to truncate the data at 32,767, which could give saturated images; 1 to divide the data by 2, which will discard probably useless precision; or 2 to subtract 32768, which will preserve the data in its entirety at the expense of having negative numbers. If a file is reopened, this is the way to set the treatment of the data to be different from the default. Using this command sets the policy for the current open file and the default for newly opened files but does not affect any other open files.

Use this command to modify the way that signed data are saved to a file that is in unsigned 16-bit integer mode. By default positive values are saved without modification, and negative values are truncated at zero. Enter 1 to have 32768 added before values are saved, which will preserve negative values. Using this command sets the policy for the current open file and the default for newly opened files but does not affect any other open files.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Below the Open menu entry is a list of the most recently used settings files. Select one of these files to open it. In this case, the settings will automatically be saved to the current file first, without requiring your confirmation. To prevent this, you could close the current settings file first.

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

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

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

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

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

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

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

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

Use this command to open the Gain Reference Policy dialog box and set options controlling which gain reference is used for gain normalization within SerialEM when several are available.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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). As of SerialEM 2.7.0, stage calibration is done over a defined distance, taking up to 11 steps if needed to cover that distance. The distances in X and Y are set with two properties, StageCycleLengthX and StageCycleLengthY, whose defaults (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 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, and the rotation angle between the lowest M-mode mag and the highest mag in LM mode.

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. Correlations often do 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 0.5 degrees.

Use this command to determine image shift offsets that will keep a feature centered as magnification is changed. This calibration is 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. One of the limitations to this kind of localization 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 the Tecnai, this is defined by default to be at �eucentric focus�; on the JEOL, a numeric value for the standard focus needs to be determined with the �ReportFocus� macro 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 is on but may be problematic otherwise.

This calibration routine is unique in that it allows you to operate the program and waits for notification that an image is aligned. It converts the first Macro button in the Camera & Macro 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 preGIF and a GIF 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 the Tecnai, 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.

Use this command to calibrate the image shift offset needed to align images from a preGIF and GIF camera. If you have such cameras, the recommended initial procedure would be to calibrate the image shift offsets on the preGIF camera, then run this command, then calibrate the offsets on the GIF 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 preGIF 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.

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

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

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.

This command allows a calibration of the C2 setting for beam crossover at each spot size. This calibration is needed in order to maintain beam intensity calibrations on both sides of crossover. 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 Tecnai alignment procedures, then rerunning this calibration will adjust the C2 settings appropriately in the beam and spot calibrations. 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.

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.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

In low magnification, use this command to record the current defocus as a standard focus that will be used when calibrating image or stage shift or taking montages. A confirmation dialog will appear. 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 magnification, the program will take the calibration from the nearest calibrated one. 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.

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 � area, binned to 256x256, with an exposure that gives 4000 counts for a 15-bit 2K camera.

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.

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

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

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.

This command will cycle through all of the available microscope magnifications and print in the Log Window the magnification index, the film magnification, and the image rotation as reported by the Tecnai (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 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 �CamerLengthTable nn�, where nn is the number of camera lengths listed.

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

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

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

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

This command will take a series of pictures with very short exposure times to determine the dead time of the beam shutter, namely the amount of exposure lost. The calibration is needed on JEOLs because the dead time can be ~0.01 sec, and this can lead to incorrect shutter time changes in routines that need to change the shutter time to reduce specimen exposure. Before running it, set the Trial exposure to 0.1 second and adjust the beam and binning to give a moderate number of counts (1/4 � � 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.

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.

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.

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.

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

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

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

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

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

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

Use this command to display the autofocus cross-correlation from pictures that are already in buffers A-C from a previous autofocusing operation. The correlation will be placed in buffer A and existing images will be rolled up one buffer.

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

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

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

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

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

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

This command toggles more complete output from focusing operations. With the option selected, the shift and drift information are printed in the Log window on every autofocus. With the option not selected, the measured defocus value and the use of a secondary peak will still be reported to the Log window, but only if it is already open.

For a list of available macro entries, see Macro Commands ; for general information about macros see About macros .

Use this command to stop a running macro at the end of a loop or the end of the macro (before repeating). In addition, when a macro is run from the Navigator, the End command can be used while the macro is stopped to terminate that macro and let the Navigator go on to acquire at the next point.

Use this command to resume a macro that was stopped with the Stop or End command. If you want to start a macro from the beginning, run it rather than resuming it. The next command in sequence will be executed if you stopped with End. If you stopped with STOP, the program will show you the line that it stopped on and ask whether you want to redo this command or go on to the next. In some cases a macro that stops because of an error condition can be resumed from the point at which it stopped; the program will resume from either the current or next command as appropriate for the type of error.

This command opens a small panel with buttons for all 10 macros. The buttons are wide enough to allow somewhat longer macro names than in the Camera & Macro Control Panel. Individual buttons will be enabled when a macro is available to run or when it is open in an editing window. If you lose the toolbar behind other windows, invoke this menu command again to raise it. The program will remember this window�s position and and whether it is open between sessions.

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

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

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

Macros can automatically repeat themselves and they can contain internal loops that are executed a specified number of times. One macro can call another like a subroutine; when the second macro finishes, the next command in the calling macro will be run. There can also be IF statements for conditionally executing some statements. Loops and IFs together may be nested to 40 levels deep and calls may be nested to 10 levels deep. A macro can also contain a statement at its end to switch to running another macro, but this is not allowed in a macro called by another macro.

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

Macros can be run from the Macro menu, from the Camera & Macro Control Panel. , or from the toolbar that can be opened from the Macro menu. In addition, CTRL F1 through CTRL F10 are hotkeys for running macros. A macro will stop running if various conditions specified in the Macro Controls dialog box are not met, such as the minimum number of counts in an image.

A macro can define a name that will be displayed in two places: on the title bar of the editing window, and in the Camera and Macro Control Panel when one of the macro running buttons is dialed to that macro. The name should be short to fit into these buttons. The name can contain one or two spaces. When a name is first added to a macro, it will appear after some action is taken with the macro.

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

Variables can be defined in macros and some arithmetic can be done in a line that defines a variable. There are three kinds of variables: ones defined on a line with an equals sign, loop index variables defined in a LOOP statement, and variables set when values are reported with almost all of the �Information Output Commands� listed below. A variable can be used in any command by putting a �$� in front of the variable name; all commands are scanned first to substitute the values of variables. Note that like commands, variables are not case-sensitive and can be referred to as �$Name�, �$name�, �$NAME�, etc.

1. A variable can be defined with an expression of �Name = value�. In general, everything in such an expression must be separated by spaces. There must be spaces between the name and the equals, and between the equals and the value. The value can consist of an arithmetic expression, which is processed and reduced to a single value (see below). Only the first value or word of text after the equals sign is stored as the value; additional text is ignored.

2. A loop index can be defined by adding an unused variable name after the number on a LOOP line. This variable will have a value from 1 through the loop count. The variable becomes undefined after the loop ends and can then be reused as a loop index or defined variable.

3. When a �Report�� command is used, the values reported are generally assigned to variables named �ReportedValue1�, �ReportedValue2�, and so on up to 6.

An arithmetic expression can contain numbers, variables that have numeric values, the 4 operators �+�, �-�, �*�, and �/�, and some function names. An expression is scanned first from left to right for �*� and �/� together; the number to the left and right of an operator are replaced by the result. Then the expression is scanned from left to right for �+� and �-� together and those operators are applied. Finally, it is scanned for some function names, currently including SQRT, SIN, COS, TAN, and ATAN, and the function is taken of the following number. If the function is ATAN2, then the following two numbers are taken as the Y and X arguments to an atan2 function that returns an angle between -180 and +180. No parentheses are allowed, so use multiple lines to do complicated computations that would require parentheses. The lack of parentheses and this order of evaluation also mean that functions will only work at the beginning of an expression, and that the function will be taken of the whole remainder of the expression. There must be a space between each value and each operator. Arithmetic can be done only in variable assignment statements and in IF statements.

An IF statement is used to start a block ending in ENDIF, in which statements are executed conditionally on the truth of the expression in the IF statement. The format of the IF statement is

where each expression may contain multiple components as long as they evaluate to a single number, and where the operator compares two values and is one of �<�, �>�, �<=�, �>=�, �==�, and �!=� (the latter two are test for equality and non-equality). If the statement is true, then following lines are executed until an ELSE is encountered, if any, at which point lines are skipped until the ENDIF. If the statement if false, following lines are skipped until either an ELSE or an ENDIF is encountered.

A common use of IF statements would be for loop control using the BREAK and CONTINUE statements. BREAK causes termination of the innermost loop being executed; the macro then continues with after the next ENDLOOP. CONTINUE causes statements to be skipped to the end of the innermost loop, but the next iteration of the loop is then run, if any.

This macro will save the current magnification and change to 20000x, then move the stage to a 4 by 4 array of positions centered around the current location and autofocus, take a Record, and save it at each position. Then it will restore the mag and the stage position. The indentation is for readability only.

This example will take pictures at a certain time interval, measure the displacement between each pair of pictures, and do this repeatedly until the displacement falls below a criterion or the limit on the number of iterations is reached. Note that important parameters are defined as variables at the top of the macro, so it would be easy for users to set parameters without having to be adept at writing such a macro.

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

	Macro Control Commands
MacroName text		Name the macro to the given text, which can include spaces
Repeat		Place at end of macro to repeat it
Loop # [variable]		Loop the given # of times until EndLoop; optionally set variable with index
EndLoop		Marks the end of the loop
DoMacro #		Switches to given macro #
CallMacro #		Calls other macro by # and returns
Call text		Calls other macro by name. Names are case-sensitive and must match exactly, including spaces
If expression		Starts a block that is executed conditionally on the value of �expression�
Endif		Ends a block started with If
Else		Inside of an If/Endif block, starts a block that is executed if the expression after the �If� is false
Break		Stops execution of the innermost Loop containing this statement
Continue		Skips the rest of the current iteration of the innermost Loop containing this statement
Delay # [units]		Wait for given amount of time. The optional �units� can be �msec�, �sec�, or �min�. If no units are given, a # <= 60 is taken to be seconds, and a # > 60 is taken to be milliseconds
Pause [text]		Print optional text and ask user whether to proceed
Echo text		Print the text to the log window

	Camera Commands
V or View		Acquire image with �view� parameters
F or Focus		Acquire image with �focus� parameters
T or Trial		Acquire image with �trial� parameters
R or Record		Acquire image with �record� parameters
L or Preview		Acquire image with �preview� parameters
M or Montage		Acquire montage
SetExposure #S #E [#D]		Set exposure and drift settling for exposure set S (0=view, 1=focus, 2=trial, 3=record, 4=preview) to the given values E and D. The change will not survive opening the camera setup dialog.
SetBinning #S #B		Set binning for exposre set S to B. The change will not survive opening the camera setup dialog.
RestoreCameraSet		Restore the first camera parameter set modified with a �SetExposure� or �SetBinning�
RetractCamera		Retracts all retractable Gatan cameras
GoToLowDoseArea area		Switches to the low dose area given by �area�, which must be one of V, F, T, R, or S. This will not work if the screen is down.

	Buffer and File Commands
S or Save [buf] [file #]		Save image in A or in buf to current open file, or to the given file number
Copy buf1 buf2		Copy from buf1 to buf2 (e.g., Copy C A)
Show buf		Display the given buffer (e.g., Show C)
SetDirectory dir		Change the working directory to �dir�, which can contain spaces.
OpenNewFile file		Open a file with the given name, which can contain spaces. The file properties dialog will be bypassed. Since the current working directory can be unpredictable, depending on whether the user has opened a file with the file chooser, it is advisable either to specify an absolute path for the filename, or use a �SetDirectory� with an absolute path.
OpenNewMontage #x #y file		Open a file with the given name for montaging, with the number of frames in X and Y given by �#x� and �#y�. The file properties and montage setup dialogs will be bypassed.
ReadFile #		Reads the given section # (numbered from 0) from the current open file
CloseFile		Close the current file.
SwitchToFile #		Make the given file number be the current open file.

	Microscope and Filter Control Commands
U or TiltUp		Tilt up by preset increment
D or TiltDown		Tilt down by preset increment
TiltTo #		Tilt to given angle
TiltBy #		Change tilt by the given amount
ChangeFocus #		Change focus by the given value in microns
SetObjFocus #		Calls SetObjFocus on JEOL with the given #, an integer increment, and reports change in focus
SetMag #		Set the mag to an actual film mag value
ChangeMag #		Change mag by given number of mag steps
SetCamLenIndex #		Set the camera length index to the given number; on the Tecnai, values higher than 30 set the index to (# - 30) in LAD mode
SetSpotSize #		Set the spot size to the given number
ImageShiftByPixels #X #Y		Change image shift by given # of pixels in camera X and Y
ImageShiftByUnits #X #Y		Change image shift by given # of IS units in X & Y
ImageShiftByMicrons #X #Y		Change image shift by given distance on specimen (X is tilt axis)
MoveStage #X #Y #Z		Move stage by micron increments in X, Y, Z (Z optional)
MoveStageTo #X #Y #Z		Move stage to given position in microns. Z is optional and Z will not be changed if it is omitted.
SetPercentC2 #		Set the C2/C3 lens strength to the given percentage
IncPercentC2 #		Change the C2/C3 lens strength percentage by the given amount
SetIntensityForMean #		Adjust beam intensity to yield the given number of counts, based on counts in the tilt-foreshortened area of the image in A
SetIntensityByLastTilt		Adjust beam intensity by the change in cosine of the tilt angle from the last tilt change
ChangeIntensityBy #		Change beam intensity by the given factor using calibrations
SetBeamShift #X #Y		Set the beam shift to the given values (nominal micron units)
SetBeamBlank #		Blank beam if # is 1, unblank if # is 0
SetSlitWidth #		Set energy filter slit width in eV
SetSlitIn [#]		Insert the slit; or if # is entered, insert if 1 or retract if 0
SetEnergyLoss #		Set the energy loss to the #
ChangeEnergyLoss #		Change the energy loss by the #
SetColumnOrGunValve #		Close column/gun valve if # is 0; open if # is 1
ScreenUp		Raise the main screen
ScreenDown		Lower the main screen
ManualFilmExposure #		Film exposure time in seconds for manual exposures, or 0 for automatic exposure (the default
ExposeFilm		Take a film exposure

	Higher Level Operations and Tasks
A or Autoalign		Align buffer A to autoalign buffer
AlignTo buffer_letter		Align buffer A to given buffer (e.g. B)
ClearAlignment		Clear the alignment shift of the image in buffer A
ConicalAlignTo buf # [show]		Align buffer A to the given buffer �buf� assuming a specimen rotation given by #. Optionally, enter 1 for �show� to show the cross-correlation.
G or Autofocus [#]		Autofocus; if optional # is -1, just measure defocus without changing
ResetImageShift		Reset image shift and move stage to compensate
ResetShiftIfAbove #		Reset image shift and realign image if image shift is greater than the given # in microns
Eucentricity [#]		Refine eucentricity, or if optional # is entered, do rough eucentricity only (1), refine only (2), both steps (3), or refine and realign (6)
WalkUpTo #		Tilt up to given angle in steps and track image position
ReverseTilt [#]		Work out backlash and realign image after reversing tilt direction; enter optional 1 or -1 to specify a specific direction for further tilts
RefineZLP		Report time of day and run Refine Zero Loss Peak procedure
NewMap		Make the current image or montage a new Navigator map
RealignToNavItem #		Realign to the current Navigator item, or to the item that this macro is being run on. For #, enter 1 to restore the microscope state to the current state after the procedure, or 0 not to
RealignToOtherItem #i #r		Realign to the Navigator item whose index in the table is #i, numbered from 1. For #r, enter 1 to restore the microscope state to the current state after the procedure, or 0 not to
TiltSeries [#s] [#e] [#inc]		Run a tilt series. Optionally, enter starting and ending angles with �#s� and �#e� (enter both or neither); if they are entered, an optional increment �#inc� can be entered too. An output file must be open, and the tilt series setup dialog must be run with either the �Setup� or �Setup Future� menu entries before running this command.
WaitForDose #d [#r]		Start accumulating dose and wait until it reaches the given #d in electrons per square Angstrom. Enter the optional #r to set how many times it reports progress while waiting (default is 10). Be sure that the screen is down and, if in low dose, that the beam is unblanked.
CenterBeamFromImage		Center beam using the existing image in the active buffer

	Information Output Commands
ReportMag		Report the current mag
ReportSpotSize		Report the current spot size
ReportPercentC2		Report the percent C2/C3 (on the Tecnai, this number matches the value in TUI) and the fractional lens strength
ReportBeamShift		Reports the beam shift in nominal micron units
ReportAlignShift		Report the alignment shift of current image in A
ReportShiftDiffFrom #		Reports the difference between the alignment shift of the current image and the given value in microns, as percentage of the value; the cumulative sum of differences in microns, and the total sum of shifts in microns. For monitoring variability in successive stage movements.
ReportAccumShift		Report sum of shifts given by ReportAlignShift or ReportShiftDiffFrom
ResetAccumShift		Reset the sum of shifts given by ReportAlignShift to 0 or ReportShiftDiffFrom
ReportStageXYZ		Report current stage position
ReportTiltAngle		Report current tilt angle
ReportFocus		Report a standardized value for the current focus setting
ReportNavItem		Report the index, stage X, Y, Z, label, and note string for the current Navigator item, or the item this macro is being run on. The index and stage coordinates are placed in �reportedValue� variables as usual; in addition, variables �navIndex� and �navLabel� are set with the index (as an integer value) and with the label string. These two variables can then be used to make filenames.
ReportOtherItem #		Report similarly on the Navigator item whose index in the table is #, numbered from 1.
ReportMeanCounts [buf] [#]		Report the mean counts of the A buffer, or of the buffer indicated by �buf�; add a 1 to get unbinned counts per second
SubareaMean x0 x1 y0 y1		Report the mean counts in a subarea of the A buffer from x0 to x1 in X and from y0 to y1 in Y. Coordinates are numbered from 0.
QuadrantMeans [n] [f1] [f2]		Report means of strips along quadrant boundaries. Optional entries are �n� for the number of pixels between strip and boundary (default 2), �f1� for the strip width as a fraction of quadrant extent (default 0.1), and �f2� for the fraction of quadrant extent to trim off the length of the strip (default 0.1). Output is labeled by quadrant number (1 to 4) and �h� for horizontal strips or �v� for vertical strips.
CircleFromPoints x y x y x y		Reports the X and Y center coordinate and the radius in microns for the circle through three points whose coordinates are given on the line as x1 y1 x2 y2 x3 y3.
ReportClock		Report seconds elapsed since last ResetClock command
ResetClock		Reset the time reported by ReportClock to 0
SuppressReports		Do not print any of these reports, just assign the values to report variables

	Setup for Work at Regular Mags Only
Tecnai		JEOL
Image shift from lowest M-mode to highest mag needed		Image shift from lowest non LM-mode to highest mag needed
Minimal requirement: one pixel size, one absolute rotation angle in regular mag range		Minimal requirement: one pixel size in each image shift range, relative rotation across each boundary, one absolute rotation angle
Recommended: Pixel size for all mags where data is likely to be taken (rotations optional)		Recommended: Pixel size and relative rotations between all mags from lowest non LM-mode to highest mag needed
	Setup for Navigator Use in LM Mode
Tecnai		JEOL
Check that eucentric focus in LM mode gives minimal image shift with beam movement, set a StandardLowMagFocus property if not		Check that standard focus in LM mode gives minimal image shift with beam movement, set StandardLowMagFocus property
Image shift for all LM mags down to lowest needed		Image shift for all LM mags down to lowest needed
Pixel size at highest LM mag		Pixel size for all LM mags down to lowest needed
Relative rotation from highest LM to lowest M mag		Relative rotations between all mags, from lowest LM needed to lowest non LM mag
Stage calibration: lowest M-mode mag, 2 or 3 mags in LM		Stage calibration: lowest good non LM-mode mag, 2 or 3 mags in LM
Image shift offsets from ~10-20K down to lowest LM mag needed		Image shift offsets from ~10-20K down to lowest LM mag needed

BiggestGIFApertureNumber	The number of the aperture that must be in place for imaging. You could image an aperture by entering its number here.
MinLowMagSlitWidth	Minimum slit width when doing low mag tasks.
SlitWideningSafetyFactor	When doing a low mag task, the slit will be opened if necessary to the minimum slit width, and intensity will be reduced by the ratio of original to new slit width times this additional safety factor.
FilterPixelMatchingFactor	Factor by which to multiply the actual, calibrated pixel size on the GIF camera when matching pixel size upon a mag change. This can be used to match field of view instead of pixel size for two cameras with different CCD sizes; e.g. one would use a value of 2 with a 1K 794 before the GIF and a 2K 795 GIF camera. It can also be used to compensate for a difference in the binning definitions of the two cameras with similar CCD size; e.g., use a value of 0.5 if there is 4K, 15-micron pixel camera before the GIF and a 2K, 30-micron pixel GIF camera.
FilterSlitInOutDelay	Delay time in DM ticks (60th of second) when inserting or retracting slit
FilterOffsetDelayCriterion	For filter offset changes less or more than the criterion, delay time in ticks is computed as change * slope1 + base1 or change * slope2 + base2, respectively
FilterOffsetDelayBase1	Base delay (in ticks) for offsets less than the criterion
FilterOffsetDelaySlope1	Additional delay per EV of offset for offsets less than the criterion
FilterOffsetDelayBase2	Base delay (in ticks) for offsets greater than the criterion
FilterOffsetDelaySlope2	Additional delay per EV of offset for offsets greater than the criterion
NoSpectrumOffset	Set to 1 to indicate that Spectrum Offset cannot be set and Energy Shift should be set instead.

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

FileOptionsMode	0 for bytes or 1 for 16-bit integers
FileOptionsExtraFlags	The sum of 1 to store tilt angles, 4 to store stage position * 25, 8 to store magnification / 100, 16 to store intensity * 25000, and 32 to store exposure dose
FileOptionsMaxSections	Default value for the maximum number of sections, which determines the amount of space allocated for the extended header
FileOptionsPixelsTruncatedLo	Number of pixels that will be truncated at 0 when scaling integers to bytes
FileOptionsPixelsTruncatedHi	Number of pixels that will be truncated at 255 when scaling to bytes
FileOptionsUnsignedOption	Determines how 16-bit data will be stored as signed integers: 0 to truncate at 32767, 1 to divide by 2, 2 to subtract 32768
FileOptionsSignToUnsignOption	Determines how signed data are written to an unsigned integer file: 0 to truncate negative numbers at 0, or 1 to add 32768

AlignTrimFraction	Fraction to trim off edges of images for autoalignment correlations
AlignTaperFraction	Fraction of image to taper to mean over for autoalignment correlations
AlignFilterSigma1	Sigma of inverted Gaussian that filters out low frequencies in autoalignment correlations
AlignFilterSigma2	Sigma of Gaussian rolloff for filtering out high frequencies in autoalignment correlations
AlignFilterRadius2	Radius at which high-frequency Gaussian rolloff starts
FocusPadFraction	Fraction to pad images for autofocus correlations
FocusTaperFraction	Fraction of image over which to taper to mean for autofocus correlations
FocusFilterSigma1	Sigma of inverted Gaussian that filters out low frequencies in autofocus correlations
FocusFilterSigma2	Sigma of Gaussian rolloff for filtering out high frequencies in autofocus correlations
FocusFilterRadius2	Radius at which high-frequency Gaussian rolloff starts
FocusMaxPeakDistanceRatio	If the highest peak in the autofocus correlation is at 0,0, the program may reject this peak and use the second highest one, if the distance from target defocus implied by the second peak position is less than this ratio times the distance implied by the peak at 0,0. Smaller values make the peak rejection more conservative, 0 disables it. The default is 3.

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

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

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

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

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

Stop	Stops macro at next command.
End	Stops macro at end of loop or repeat.
Resume	Resumes macro at place where it stopped.
Toolbar	Opens a toolbar with buttons for all 10 macros.
Controls	Opens dialog box to set conditions for stopping a macro.
Edit 1, 2, ...	Opens a window for editing a macro.
Run 1, 2, ...	Runs a macro.

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