SerialEM Help Index. ABC Amber HLP Converte r T rial version,

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1 SerialEM Help Index Genera l Topics Introduction to SerialEM Mouse and Keyboard Controls Control Panels Image Acquisition Acquiring Tilt Series Montaging Low Dose Mode Energy Filtering Using the Navigator Settings Files Macros Macro Commands Setting Up SerialEM Property File Entries Commands File menu Settings menu Camera menu Calibration menu Focus menu Macro menu Tasks menu Tilt Series menu Process menu Navigator menu Window menu Help menu Control Panels Buffer Status panel Buffer Control panel Image Display Control panel Microscope Status panel Tilt Control panel Camera & Macro Control panel Image Alignment & Focus panel Low Dose Control panel Montage Control panel Filter Control panel Dialog Boxes File Properties dialog Camera Setup dialog Gain Reference dialog Gain Reference Policy dialog Montage Setup dialog Tilt Series Setup dialog Tilt Series Resume dialog Tilt Series Back Up dialog Tilt Series Extra Output dialog Tilt Series Autostart Policies dialog Navigator window Navigator Acquire dialog Macro Control dialog Macro Editing window Page 1

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3 Introduction to SerialEM SerialEM presents an integrated environment for image acquisition, display, and storage, as well as for the control of the Tecnai microscope needed to acquire montaged images and tilt series. You can acquire images from Gatan cameras using parameters that you set from within SerialEM. Images can be zoomed, panned, and compared with each other using controls similar to those in Imod. Successive images are conveniently saved to a single MRC file, ready for display in Imod. The Tilt Series Controller will acquire a tilt series automatically using a prediction algorithm to minimize acquisition time and specimen exposure. A low dose mode can be used to perform focusing and tracking away from the area of interest. SerialEM can also automatically capture a montage of overlapping frames, and acquire a montaged tilt series. The program also contains a macro feature for programming repetitive actions. Controls: SerialEM is controlled through its menus, control panels, and hotkeys. Menus contain commands related to a topic, as well as some entries to set parameters related to that topic. The control panels provide status information, buttons for performing all frequently used operations, and buttons for setting parameters related to the particular panel. Here is some general information about Control Panels. Hotkeys are summarized in the section on Mouse and Keyboard Controls. Buffers and display windows: Images are initially displayed in a main image display window. Images are kept in a set of buffers, and the main display window can show an image from any one of these buffers. Similarly to Imod, you can riffle through the images with PageUp and PageDn. The buffers are referred to by letter. Images from the camera are always placed in buffer A, the first buffer. The program is typically set up to roll images through the first three buffers. In other words, when a new image comes in, the existing image in buffer A is moved to buffer B, the image previously in buffer B is moved to buffer C, and the image in buffer C is lost. It is also possible to copy an image to a new, free-standing window, which could be useful if you want to look at two images side by side. Camera control: The camera controls are patterned after those available in Digital Micrograph, but there are some important enhancements that provide more flexible control of the specimen exposure, make it easier to select subareas of the camera, and allow control of whether a dark reference is taken. General information about image capture is available in the section on Image Acquisition. File storage: When images are saved to a file, successive images are typically stacked into a single file in the MRC format. The pixel size is set in the header, and tilt angle and other information can be stored in the extended header area. The file header is maintained after every image is stored, so that the file will be readable if something goes wrong. Image alignment: Alignment of images is a key component of data collection. SerialEM provides a linkage between the shifting of an image in a window, which is referred to as an alignment shift, and the physical realization of this shift using the image shift feature of the microscope. This means that if you want to center a particular feature in the camera frame, you can impose an alignment shift on an image that has already been acquired, simply by dragging it with the right mouse button. The microscope image shift is then changed by the right amount so that the next image that you acquire appears in the desired place. Two images can also be aligned to each other by cross-correlation. This is referred to as autoaligning. The image in buffer A is correlated to a reference image in another buffer. Before doing this correlation, the program will stretch the image taken at a higher tilt angle to give a better match to the other image. Autofocusing: SerialEM determines the defocus of the specimen by the standard technique of measuring how much the image moves when the beam is tilted. When the specimen is in the focal plane, its image does not move; and the farther it is from focus, the bigger the beam-tilt induced movement. For this to work correctly, you need to align the beam tilt pivot points properly, a standard step in the Direct Alignments used to tune the microscope. Be sure that the specimen is at the eucentric height and at minimum contrast focus when you tune the pivot points. They do not need to be perfect, but do need to be close. Measurement of defocus is typically done by taking a picture with positive beam tilt, one with negative beam tilt, then another with positive beam tilt. Having three pictures allows the program to compensate for drift. Once the program has measured the defocus, it can change it to achieve the level of defocus that you specify. This procedure is called autofocusing. Page 3

4 Mouse and Keyboard Controls Mouse actions in the image display window: Left mouse button click: place a marker point in the image and print the coordinates and value in the middle panel of the status bar. Several procedures make use of the position of a marker point. Ctrl left mouse button click: zoom up. Ctrl right mouse button click: zoom down. Left mouse button drag: Holding the left button down and moving the mouse will pan the image, if it is zoomed bigger than will fit in the window. Right mouse button drag: Holding the right button down and moving the mouse will change the alignment shift of the image. If the image is in buffer A, this will also change microscope image shift so that the next image acquired will match the image with this alignment shift. If there is a marker point on the image being aligned to, its position will show up in red. There is an option available to have particularly large moves with the right mouse button move the stage instead of change the microscope image shift. Shift - right mouse button drag: Shifting the image in buffer A while holding the Shift key down will result in the stage being moved, rather than microscope image shift being changed. Hotkeys: -/_ Zoom an image down =/+ Zoom an image up. Ctrl A Autoalign Ctrl B Toggle Blank beam when screen down in Low Dose control panel Ctrl D Tilt Down Ctrl F Acquire image with Focus parameters Ctrl G Autofocus Ctrl H Halt camera acquisition Ctrl I Image information min/max/mean and SD. Ctrl L Acquire image with Preview parameters Ctrl M Start a montage Ctrl O Open old image file Ctrl P Open camera parameter dialog box Ctrl R Acquire image with Record parameters Ctrl S Save image to file Ctrl T Acquire image with Trial parameters Ctrl U Tilt Up Ctrl V Acquire image with View parameters Space bar Stop or restart continuous image acquisition. Esc Stop camera acquisition and any running tasks. F1 Open context-sensitive help when the mouse is over a menu item or a dialog box. Shift-F1 Will allow you to click on an item and open help for it. Ctrl F1 Run macro 1 Ctrl Fn Run macro n, n = 1 to 10 Shift B Binned FFT Shift F FFT Shift L Toggle live FFT (for continuously acquired images) Shift R Read an image from file Ctrl-Shift R Resize the main image window to fill the SerialEM frame. The keys in the 6-key cluster above the arrow keys do the following: PageUp PageDn Home display the previous occupied buffer (a lower letter). display the next occupied buffer (a higher letter). display buffer A, or the first occupied buffer. Page 4

5 End Insert Delete display the last occupied buffer (highest letter). display the buffer that is being autoaligned to. display the buffer that is being read into from file. The four arrow keys will pan the image by small steps if it is zoomed bigger than the window. Page 5

6 Image Acquisition An image is captured from the CCD camera with a sequence of 3 steps: clearing the CCD chip, exposing the camera to the beam and integrating charges for some period of time, then reading the image off of the chip. The readout is done line by line, so it is often very time-consuming. The clearing time is also substantial because the chip is cleared by shifting accumulated charges to one edge. Clearing times are about 0.7 to 0.9 second for 2K Megascan or 4K single-port readout Ultrascan cameras, 0.25 second for a 4-port readout Ultrascan, or very short for some current 1K cameras. It is important not to have a beam on the camera during the clearing time, because then charges will build up as they are being removed, adding a ramp of intensity to the image that is subsequently acquired. Many plastic-embedded specimens show a transient shift of the image when the beam is turned on to take an exposure; this drift produces a smeared image. To take good images, you need to use drift settling, which provides an initial exposure of the specimen to the beam just before the image is acquired. This initial shift is worse on slot grids than on mesh grids and tends to be worse at high tilts. It seems to depend on the total beam hitting the sample rather than the brightness of the beam in the area of interest. This means you should work with the largest spot size that gives you enough beam. Exposing the specimen to the beam before the exposure without exposing the camera during its clearing time requires the use of at least two shutters, one below the specimen and one above. The traces below show how DigitalMicrograph manages the two shutters in standard shuttering mode with the alternate (beam) shutter normally closed. A trace goes high to indicate when a shutter is open or beam is on the camera. Although this shuttering provides some pre-exposure, there are two problems here. First, the amount of pre-exposure is fixed at the clear time, which could be too short or longer than needed. Second, the beam is left on the specimen during camera readout. (Note that this occurs only when using the shuttering mode that provides pre-exposure and does not happen if you are just using the beam shutter for low-dose exposures.) DigitalMicrograph does provide a way to increase the drift settling, but this has problems of its own. As shown in the following traces, the extra settling is implemented by keeping the beam on the CCD during the clear time. This introduces an intensity ramp in the image, which is eliminated by having the beam on for the equivalent time during the dark reference. These ramps use up some of the dynamic range of the camera, but worse than that, the dark reference contains image features and will become invalid when exposure or specimen location changes. Page 6

7 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. Page 7

8 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. Page 8

9 Acquiring Tilt Series The basic steps involved in acquiring a tilt series are quite simple: tilt by an increment, adjust image position to keep features of interest centered, adjust focus to maintain a desired level of defocus, and acquire and save an image. However, several factors make this process considerably more complicated. First, it is highly desirable to skip some of these steps as often as possible, because of the time and specimen dose required to perform them on every tilt, yet it is not trivial to skip steps without jeopardizing the quality of the tilt series. Second, the need to track image position with a lower magnification image when working at a high magnification, or with an image away from the area of interest when trying to minimize dose, makes this operation more complex. Third, things do go wrong that require manual intervention, and the program must have the flexibility to allow for a variety of interventions. The part of SerialEM that manages tilt series acquisition is called the Tilt Series Controller (TSC). It deserves to be thought of as an entity in its own right because it takes control of many aspects of program operation. For example, you will not be able to tilt, copy images to certain buffers, or save images directly while the TSC is in control you can do these things only through the TSC. When the TSC is running, buffer usage is standardized as follows: images roll from buffer A through buffer C as new images are acquired; buffer D is the primary buffer for autoalignment, buffer E is a secondary buffer for aligning low magnification images or Trial images in low dose mode; and buffer F can be read into from a file. Copying to buffers E and F is disabled. You can suspend a tilt series to change the image alignment or focus. See the help for the Tilt Series Resume dialog box for more details on what options are available when you do this. You can also back up to previous tilt angles; see the help for the Tilt Series Back Up dialog box for more details on this process. Minimizing tracking errors Running a tilt series continuously from one extreme tilt to the other can result in the area of interest not being well centered at zero tilt. This can happen either because of inaccurate tracking from high to low tilt, or because it was difficult to recognize the correct center position at the starting tilt angle. SerialEM has several features that can help prevent this problem. One is the Walk up procedure, which allows one to start at zero tilt (or a higher tilt if desired) and tilt up to the starting angle in a series of coarse increments with tracking images that are successively aligned to each other. This could be particularly useful when it is hard to recognize the features of interest at high tilt. You can activate the Walk Up procedure yourself (from the Tasks menu) before you start the TSC, or you can activate the TSC at a low tilt angle and have it walk up to the starting tilt angle. The second feature that may help tracking is called an anchor. This is an image taken at around 40 to 50 degrees that the TSC can use as a reference when the tilt series reaches that angle. The angle should be low enough so that tracking is likely to be accurate from that point downward, but high enough so that uncorrected Y-axis errors above that point are unlikely to cause data loss in the reconstruction. (Note that because the region of interest is foreshortened in the Y direction at high tilt, alignment errors can be quite high in Y without losing any of the region of interest.) Angles in the range of degrees should satisfy these criteria. If you are starting the TSC at a low tilt angle and having it walk up to the starting angle, you can have it leave an anchor at an appropriate angle. If you are going to high tilt before starting the TSC, you can also specify an angle to leave an anchor at when you use the Walkup & Anchor procedure. The fallback feature for fixing a tracking error is manual intervention. Simply stop the series with the End button. It will stop with a Record image in Buffer A. Shift this image to the desired alignment with the right mouse button. Then select the Resume option, check the box Use image in buffer A as reference for alignments in the Tilt Series Resume dialog box, and push the GO button. Starting a tilt series The TSC can be started either at a low tilt angle or at the starting tilt angle. You can safely start at low tilt if you are fairly confident that: The camera parameters (exposure time and drift settling) are suitable for getting good pictures at Page 9

10 high tilt. Autofocusing will work well enough to find focus at high tilt. The beam can be adjusted automatically to give the desired counts in the image without becoming too small and encroaching on the camera area. If you have doubts about any of these factors, you should go to high tilt before starting the TSC, and make sure that images look good, that the specimen is focused, and that the beam can be adjusted properly. These conditions may be satisfied if you have already done one tilt series on the specimen, in which case letting the TSC start from low tilt would save some effort. Here is a sequence of steps for starting a tilt series: 1. Decide if you are going to start at low tilt or go to high tilt before starting the TSC. 2. If you are going to high tilt before starting the TSC, refine eucentricity. Select the Refine and Realign command if you have already centered your region of interest and plan to use the Walk Up procedure; otherwise Fine Eucentricity is sufficient unless the magnification is high enough that you might lose your selected area. 3. Go to high tilt. Consider using Walkup & Anchor to save an anchor, as described above. 4. Make sure that the exposure settings for Focus, Trial, and Record give images without drift. 5. Do autofocus and see if it gives a well-focused Record image with the best focus in the center of the field. If not, you can use the Move Focus Center command in the Focus menu to move the center of focus to the middle of the field, or you can vary focus by hand. Once focus is found, you should run the Measure Defocus command in the Focus menu and use the reported value to set the target defocus. 6. Set the beam intensity to give the desired level of counts. You can do this by hand or use the Set Intensity command in the Tasks menu. This can be done with either a Trial or a Record image in buffer A. When you select the command, the message in the dialog box will show the mean counts that a Record image would give with the present beam intensity. You can enter either a new number of counts, or a factor by which to change the intensity. If you change the number by much (more than 10-20%), you should take another image and look at the beam on the screen to make sure it is not too small. If you cannot comfortably achieve the desired value, just adjust the beam so it is large enough. You will be able to select an option to reach the desired target number of counts at a lower tilt angle, and the program will reach this target as fast as it can without making the beam smaller than this initial setting. 7. Take a final Trial image and center the features of interest. 8. Open the Tilt Series Setup dialog box in the Tilt Series menu and check the entries from top to bottom, paying particular attention to the following: 9. Check the starting and ending angles and angular increment. 10. Make sure the delay time after tilting is appropriate for the holder and tilt increment (small increments need less delay time; heavy holders need more). 11. Decide whether you need low magnification tracking. This is recommended for magnifications above 50,000, and might be needed at a somewhat lower magnification if you are starting at a particularly high tilt angle (say, above 70 degrees). 12. Make sure the limit on image shift is appropriate for the objective aperture size and other conditions. On the 300 KV Tecnai, operating at a relatively low magnification, montaging, and using low dose may all require a smaller limit on image shift, perhaps as low as 1 micron. When the limit is reached, the TSC will automatically reset the image shifts, move the stage to compensate, and recover from the physical disturbance caused by this movement. 13. Make entries for beam intensity control. Checking Do not increase intensity above value for first Page 10

11 saved image is almost always a good idea. If you are starting at high tilt and needed to set the beam to give less than the desired target number of counts, check Keep intensity below current value (use if intensity already set up). This will achieve that target as soon as possible without overconstricting the beam. On the other hand, if you are starting at zero tilt and know that you need to reduce the number of counts at high tilt, fill in the desired target number in the Set intensity to keep mean counts of Record images at text box, check the Taper counts down to option, and put your reduced target for the highest tilt in the text box there. 14. If you have had difficulty with autofocus at high tilt, you can select the Focus every time above option and fill in the desired angle above which you want to focus on every tilt. 15. If you have a centered image in buffer A, select the option to Align to image now in Buffer A. 16. If you are starting at zero tilt, you should select the option to Refine eucentricity first unless you have already done this. 17. If you are starting at high tilt and have acquired an anchor image on the way up, select the Use mid-tilt anchor option and then use the up or down arrow if necessary to indicate which buffer contains the anchor (it should be in the right place if you used Walkup & Anchor ). 18. Under Tracking control parameters, you typically need to consider only the first line, which controls whether Record images will be repeated if they are not centered well enough. If you never want this to happen, unselect the option. The percentage here will determine the width of the area near the edge of your reconstruction that is degraded because some images fail to contribute data to it. A value of 5% is probably reasonable for most uses. If the structures of interest nearly fill the camera frame at low tilt, then you might want to reduce this to 2.5-3%. If you are working at very high magnification, you might want to raise the value or unselect the option. 19. If you are working at high magnification where tracking will be relatively less precise, you may need to increase the value for Get tracking image when error in X/Y prediction is > to avoid excessive tracking images. You may also need to increase the criterion for Get new track reference if Record alignment differs. After you start the tilt series, the TSC will perform a number of preliminary actions, which may include finding eucentricity, walking up, finding focus, getting a first reference image, and adjusting intensity. Also, the program will use the Reverse Tilt procedure to eliminate tilt backlash, so there is no need for you to do this yourself. This procedure involves tilting up and back by about 3 degrees, with lower magnification pictures taken before and after to track the potentially large shift in position that can occur. If there is pole touch in this process, the program will try to compensate for this by taking lower magnification tracking images during the first few tilts, until the backlash is worked out. How predictions work The TSC minimizes the number of tracking and focusing positions by predicting where the specimen will be after each tilt. It predicts position separately in the X and Y directions (along and across the tilt axis) and uses image shift to compensate; it predicts the position in Z and changes focus to compensate. At the beginning of the tilt series, the information needed to make reliable predictions accumulates gradually. After two tilts, there is enough information to make simple extrapolations, but not to know how accurate they are. With data from three or more tilts, the program can assess the accuracy of a prediction from how well the positions fit a straight line. In addition, if predictions were made on the previous tilt, the program can determine how far off they were. Thus, at a given tilt angle, there are two kinds of prediction errors available: the computed error of the prediction for the next angle, and the actual error in the prediction for the current angle. Only when both of these errors are low enough will the TSC rely on a prediction and skip a tracking or focusing step. The accuracy of the prediction generally increases as more data become available, but only as long as the positions change regularly. Because the specimen and stage do not behave ideally, it is necessary to restrict the positions used for the predictions to those from the most recent tilts. For each axis, there is a maximum tilt range over which positions will be fit (a parameter that can be set in the.) Moreover, the TSC Page 11

12 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. Starting Multiple Tilt Series Automatically 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. In method 1, you would have a macro such as the following: MacroName TSeries SetDirectory E:\mast\ptk ReportNavItem OpenNewFile prometa-$navlabel.st Call Cooker Call Center TiltSeries TiltTo 0 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 Page 12

13 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. For method 2, you could use two macros such as this: # Macro to run series of tilt series MacroName TSeries SetDirectory E:\mast\ptk rougheucen = 1 restorestate = 0 item = 2 Call PrepTS TiltSeries Item = 4 Call PrepTS TiltSeries # Macro to prepare for tilt series at one location MacroName PrepTS TiltTo 0 ReportOtherItem $item If $rougheucen!= 0 MoveStageTo $reportedvalue2 $reportedvalue3 Eucentricity 1 Endif RealignToOtherItem $item $restorestate OpenNewFile prometa-$navlabel.st Call Cooker Call Center 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 Page 13

14 abandoned. 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 macro 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. Page 14

15 About Montaging Montaging allows one to capture an array of multiple, overlapping frames automatically using either electronic image shift or stage movement. Each such frame is called a piece. Pieces are numbered and referred to by their position in X and Y within the montaged image (numbered from 1) and by their section number or Z value (numbered from 0). For example, in the first set of images making up a 2 by 3 montage, piece 2, 2 is the middle piece in the right column, and the Z value would be 0. All pieces in a montaged image have the same Z value. Montaging can be initiated in several ways: by selecting 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 Page 15

16 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. Page 16

17 About Low Dose Mode The essence of low dose mode is that focusing and tracking operations for a tilt series are done in a location separate from the area being recorded, to spare that area from unnecessary beam exposure. In order to provide accurate focusing and tracking, these separate areas must be displaced along the tilt axis from the center of the recording area. SerialEM s low dose mode provides 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. In low dose mode, numerous features of SerialEM operate differently. Tasks that use low magnification tracking (finding eucentricity, reversing tilt, and resetting image shifts) will automatically use View images. Walking up will use images of the Trial area. Autoalignment operates with two autoalign buffers. A Record or Preview image will be aligned to the image in the first autoalign buffer, whereas a Trial or Focus image will be aligned to the second autoalign buffer. The Align to button in the Image Alignment & Focus control panel will indicate which are the two autoalign buffers. Autoalignment will work with the beam constricted inside the camera field because autoalign will detect dark areas in the corners of the image (namely, areas outside the beam) and use only the largest rectangle inside these areas for alignment. The Tilt Series Controller uses D and E as the two autoalign buffers. It uses images of the Trial area for tracking, but it also maintains a reference image from the Record area, so that each new Record image is aligned to that reference and then replaces it. If you choose to adjust intensities automatically during a tilt series, only the intensity of the Record area will be adjusted. You can set the properties of a low dose area by making it be the current area then adjusting its features. The current area and its beam properties are displayed on the second line of the Low Dose control panel. Page 17

18 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. Steps for Initially Setting up Low Dose It is somewhat difficult to get low dose set up initially, but following a set procedure may be helpful. 1) Before going to low dose mode, set the magnification and beam strength that you will want for the View area. 2) Turn on low dose mode and take a picture of the View area. 3) Turn on Continuous update to adjust the View area properties further. Be sure to set any View-area-specific GIF settings. 4) Copy View properties to the Record area with the R button in Copy current area mag & beam radio group. 5) Take a Preview or Record picture to go to the Record area, then turn on Continuous update. 6) Adjust the magnification for the Record area, and any specific filter settings. 7) Adjust the beam strength by taking a Preview or Record and selecting Set intensity in the Tasks menu. Enter the desired number of counts in a Record image. If there is a big change, this may take 2-3 iterations. 8) If the beam is too small, reduce the spot size; if it is too large (i.e., you are concerned about extra Page 18

19 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 ) Make sure Keep Focus and Trial identical is selected and copy Record properties to the Trial area with the T copy button. 11) Select the radio button to define the position of the Trial area. 12) Take a View picture. Click with the left mouse button to define a position for the Trial area. Note the Maximum area separation and click again to get a sufficiently positive separation, or just type in a position. 13) Take a Trial picture. Adjust area-specific filter settings (e.g., slit out). 14) Here it is essential to get the beam constricted to just the Trial area and centered on it. You can do this either by adjusting brightness and position blindly or by using the Set Intensity and Move Beam operations in the Tasks menu. When using Set Intensity, note that changing intensity by a certain factor should change beam diameter by the square root of that factor. Also note that your changes will not be accurate when the beam does not fill the camera field. In general, the procedure is: a. Constrict the beam enough so that you can see enough of its edge to be able to center it. b. Change spot size if necessary to get close to the desired intensity. c. Center the beam. d. Expand the beam until it just circumscribes the area. If you are restarting with some existing parameters, you would not do all of these steps. You would probably take a View and adjust its properties if necessary (steps 2 and 3), take a Record and adjust its properties (steps 5-9), then adjust the Trial/Focus area (steps 11-14). Page 19