Multiple Record Setup Dialog
This dialog allows you to set parameters for taking multiple Record images with image shift used to center on a series of positions. The positions can be in a circular pattern around the current position, which would generally be used for taking multiple shots within a hole, or in designated positions relative to the current position, which would generally be used for taking shots in multiple holes. Images can also be taken in two concentric rings of positions around the center. Both within-hole and between-hole images can be taken. The parameters are used in two ways: when acquiring the images with the 'MultipleRecords' script command, and when displaying the positions as a pattern of circles representing the beam on an image (e.g., a lower-magnification map). The latter display occurs when this dialog is open, and also when the 'Show Acquire area' checkbox in the Navigator dialog is turned on and the display is enabled with the Show Shots when Show Acquire option in the Navigator - Montaging & Grids submenu. When the relevant parameters here are changed, these changes will appear immediately in the display.
It is important to realize that if the image areas are being displayed on a Low Dose View image with a large defocus offset, the positions and sizes will be misleading unless the effects of defocus on magnification and image shift are calibrated.
If the stage is tilted, the program can respond in two ways. First, if the tilt angle is above 10 degrees, it will compensate for the differing heights of the positions by changing defocus; the change is determined from the tilt angle and the image shift at each position. The compensation is imperfect because the specimen may not be level at zero degrees, making the effective tilt angle when tilted different from the nominal one. Second, if the image shifts of multiple holes are defined at one tilt angle and acquisition is done at another angle, the program will adjust the positions appropriately. This adjustment is also imperfect for large differences in tilt angle but may be useful in some cases.
Saving Accurate Image Shift Values for Holes
If you have used the hole finder to mark hole positions, the best way of getting image shift values may be to use the Use Last Hole Vectors button. Since these vectors are stored as stage positions, they can be accurately converted to image shift positions at the Record magnification even when they were detected on a highly defocused View image (provided that the High Defocus Magnification calibration has been done). Otherwise, there are several possible methods of setting the image shift values for the holes, ranging from centering holes in an image where they are all visible (e.g., low dose View), through centering consistent positions at a magnification still lower than the Record mag, to centering positions at the Record mag itself. Your choice depends on how accurate the positioning needs to be (more accuracy is needed for holes at more distant image shifts or if multiple shots are to be taken inside the holes), and on how well image shift positions correspond between the Record mag and the mag where they are defined (a constant offset is not a problem, scaling or rotation is). You can try an easier method first and see if it is adequate for your situation. See the section at the end of this page on how to assess that point .
The goal is the save image shifts at consistent positions, not necessarily the ones at the center of the holes, because only the differences between shifts matter. It is just as good to save the shift at the edge of each hole, if a corresponding point on the edge can be picked accurately enough. Here are two methods of positioning along a diagonally oriented part of a hole edge:
All of these methods require the Navigator to be open and suggest that you add a group of points on an image at the locations where you want to save image shift values. This step is helpful if you have more than a 2x2 pattern of holes, to avoid losing track of which hole you are on. You should add those points in the order in which their positions will be saved (see 'Save Image Shift Values at Holes' below.) When you press the button to start saving image shift values, the Navigator buttons on the left change, 'Go To XY' and 'Go To Marker' change to 'IS to XY' and 'IS to Marker' respectively. They can be used to move to marked positions with image instead of stage shift. The same operation can be done with the 'IS to Nav Pt' button in this dialog.
1) Centering holes at a lower mag. In order for image shift positions to correspond well enough between this mag and the record mag, this operation must be done near focus (within about 20 microns), unless there are calibrations for both magnification and image shift at high defocus. The message line below the buttons for recording image shifts will indicate whether defocused image may work. Assuming that you use View images, the steps are:
This method can be accurate to within 1% on a scope with good correspondence between View and Record positions, but can be off by a lot.
2) Picking consistent positions at an intermediate mag where it is still easy to find the hole. Even if you cannot use the View mag, you will probably get accurate positions at some mag closer to the Record mag (e.g., half the mag), because image shifts are likely to be consistent at higher mags. You will probably be able to use this method without changing the View defocus offset, but you will need to change the Record mag. The program will save the Record Low Dose parameters when you start recording positions and restore them when you stop, as long as you are still in Low Dose mode with the same camera selected. The steps are:
3) Picking consistent positions at the Record mag. This method is guaranteed to give accurate image shift values, as long as you can define consistent positions. If you think that you can position edges well enough, then the procedure is the same as just described, except that you do not need to change the Record mag. However, if you have difficulty finding the holes, you may need to reduce the View defocus offset close to zero. This will likely throw off the View shift offset, and you might need to adjust that too.
An alternative to positioning edges is to take a Prescan montage at the Record mag big enough to show the whole hole, and center that. This may be reasonably fast, especially if you use continuous mode with a K2/K3 camera. The steps are:
The Dialog Controls
Do multiple Records within each hole
Select this checkbox to take images in a circular pattern. The relevant controls will appear when it is selected. The property 'MultiInHoleStartAngle' can be used to control the angles of the positions. In the absnce of that property, if the specimen is tilted above 20 degrees, the first off-center shot will be done on the tilt axis, so that you can take up to three shots along the tilt axis in a hole. Otherwise, the first shot is taken along the camera X axis, or along the short axis of the camera for a non-square camera, which may allow two or three shots to fit along that axis when they would not along the long axis.
Number of shots in main ring around center
Select a value between 2 and 18 for the number of Records taken in off-center positions in the first or only ring of positions.
Distance from center to main ring of shots
Distance from the center position to the first or only ring of off-center positions, in microns. This distance must be at least as big as the beam diameter to avoid exposing overlapping areas in more than one Record shot. If the multi-shot circles are being displayed, press tab or Enter to have the image redrawn after entering a new distance.
Do second ring of shots
Select this checkbox to take Records in a second ring, which will be taken after the first ring but can be either inside or outside it. Use the spinner to select the number of shots in the second ring, and enter the distance from the center, in microns, in the edit box.
Shot in center
With these radio buttons, you can include a shot in the center of the pattern, which should be possible with 6 or more off-center shots. The centered shot can be taken either before or after the off-center shots. Although taking it may involve one more delay for image shift settling, it is reported that this shot will have less drift when taken after the others.
Do Records in multiple holes
Select this checkbox to take Records in either a rectangular or hexagonal grid of positions or a series of arbitrary, 'custom' positions around the current center position. The relevant controls will appear when it is selected.
Multiplier of usual delay after shifting to hole
SerialEM has a built-in settling time after image shift that depends on the size of the shift; it is probably inadequate at high magnification. The factor entered here increases this settling time when shifting between holes; the extra delay time described below is still added to the settling time. This separate entry allows you to have an adequate settling time when moving between holes without having to make the extra delay for shots within a hole bigger than necessary.
Regular pattern of holes: n by n
Use the two spinners to set the number of positions in a rectangular pattern. These values must be set before recording the image shift values at the corners. Once the spacing of the pattern is defined, the spinners can be changed to acquire more or fewer holes at that spacing. SerialEM will generally acquire the number of positions specified by the current setting in the dialog (or the last setting there when it was open). There are two important exceptions to this: 1) If Edit mode is turned on in the Navigator, then Shift-Left double-click with the mouse sufficiently close to one of the positions in the array around the current item will remove that position. After that, the size of the array becomes a permanent property of that item, and it will be both displayed and acquired as an array of that size (minus the removed position(s)) regardless of changes in the selection in this dialog. 2) The Multi-hole Combiner also makes the pattern to use a permanent property of each item that it creates.
If for some reason you are not using the Multi-hole combiner, you can do the following to avoid having to remove many points from a large array when marking points at the edge of a grid square after filling the rest of the square:
Select this option if the grid holes are in a hexagonal pattern. The first spinner now sets the number of hexagonal rings to acquire around the center point and the second spinner disappears.
Omit corners of 3 by 3 array (cross pattern)
This option allows one to acquire a cross-shaped pattern of 5 holes, which will involve less image shift than a full 3 by 3 array while still allowing one to center the marked point on a hole instead of between holes. It may also be useful when marking holes at the edge of a grid square after filling the rest of the square with a larger pattern, as long as one removes at least one position from the cross pattern.
Use custom pattern
Select this checkbox to take images in an arbitrary set of positions specified by shifting to a center location, which is not acquired, then to each acquisition position. A custom pattern can be used when you want to acquire at positions that are not regularly and symmetrically arranged around the marked Navigator point.
Save Image Shift Values at Holes
Press For Corners of Regular Pattern to start setting the image shifts needed for a regular pattern, or For Custom Pattern to start setting the shifts for arbitrary positions. Press Step To and Adjust IS to visit the corners of a regular pattern, or all of the positions in a custom pattern, and adjust the image shift there, and save the new shifts. In the latter case, when in Low Dose mode, the Step and Adjust IS Setup dialog will open to allow you to set the conditions for imaging. After pressing one of these buttons, the label below the third line of buttons will summarize what to do next.
To set the corners of a regular rectangular pattern, you will save to image shifts at 4 hole positions, even if one of the spinners is set to 1. Start at one corner of the pattern and save the position. Then shift by the needed number of holes corresponding to the value of the first spinner (i.e., move by 2 holes if the value is 3) and save that position. Shift by the number of holes indicated by the second spinner, save, and shift back by the number of holes along the first axis. If one of the spinners is set to 1, the program will still expect you to shift by one hole along that axis.
To set the corners for a hexagonal pattern, you must shift to each of the 6 holes in the ring specified by the spinner.
To set the positions in a custom pattern, shift to a center position for the pattern and save the shift. When taking multiple Records, this position will not be acquired at, but all the shifts will be done relative to it. Then shift to the first point to be acquired and save that position. Repeat until the last point and press End Pattern.
When adjusting existing image shift values, new image shifts are recorded at the same sequence of positions as when first saving them. You should start out positioned on the center of the pattern. If a regular pattern is selected (i.e., if Use custom pattern is not checked), press Step To and Adjust IS, and the image shift will be set to reach the first corner of the pattern. Press Save Image Shift after adjusting the image shift (or not), and it will move on to the next position, eventually going to all four corners. If a custom pattern is selected, press Step To and Adjust IS, then adjust the centering on the origin of the pattern if necessary. Press Save Image Shift to store that reference location, and it will move to the first position to be recorded. Continue through the rest of the positions to be recorded. When adjusting image shift, the magnification will be set to that at which image shifts were previously defined, and in Low Dose mode the Record magnification will be set that value as well as long as it is in not closer to the View magnification. However, you are free to change the magnification at which you adjust the shifts.
See above for instructions on how to save sufficiently accurate shift values with these methods.
IS to Nav Pt
Press this button to move to the location of the current Navigator point with image shift and then select the next point in the table. This saves you from pressing 'IS to XY' and selecting the next point in the Navigator window, extra steps where you can lose track of whether you have changed the selection there. Press this button on first starting the saving operation and immediately after each 'Save Image Shift'. The first time that you do so, the program looks at how many points are in the current group and which one is selected. If the first one is selected, or if you are defining a regular pattern and there are 4 points, it will image shift to the first point in the group and select the next one. Otherwise, it will ask you if you want to start from the beginning and work forward, or start at the end and work backward (which is not going to work for a custom group unless you marked the central point at the end), or simply leave the selected point alone.
Save Image Shift
Press this button to save the current image shift at the next corner in a regular pattern, or the next spot in a custom pattern.
Press this button to end the entries when defining a custom pattern. It can be pressed either after saving the last position, or without explicitly saving it. The current image shift will be saved if it is different from that of the last saved point.
Press this button to stop entering or adjusting shifts and throw away all entries.
Use Last Hole Vectors
Press this button to convert the vectors from the last hole-finding operation to image shift values for a regular pattern. This operation is equivalent to using the 'UseHoleVectorsForMulti' script command. The program will ask if you are sure you want to replace the current image shift values, in case the button was pressed accidentally. You can press 'Yes Always' to avoid that confirmation for this session of the program. The tilt angle at which the holes were found must match the tilt angle last used to define the vectors (but in fact there is no compensation for tilt so holes need to be found at zero tilt.) You must be in Low Dose mode for the button to be enabled.
Step To and Adjust
Press this button to step to each of the positions of a defined pattern and adjust the image shift there. See detailed instructions above.
Save Record image
Select this option to have the Record image saved in the current file. The option is disabled and its setting is irrelevant if an early return is selected for all Records with no returned image. However, if any images are returned, they will all be saved as of SerialEM 4.0.
Early returns for K2/K3
This group of controls appears when there is a K2 or K3 camera available and allows for SerialEM to resume operations as soon as possible after acquiring an image with saved frames, the same as is accomplished with the script command 'EarlyReturnNextShot'. The radio buttons allow you to select to have an early return only on the last shot, on all shots, or on all shots except the first one. The latter option allows you to assess quality (e.g., from an FFT or from frame alignment results) on the first shot without having it quickly replaced by another. If post-exposure actions are enabled for the camera (namely, if Post-actions in the Camera menu is not grayed out and is selected), then image shift to the next position will occur soon after the exposure is finished for each shot, regardless of whether early return is selected, and it is slightly more efficient not to do an early return except on the last shot. If post-actions are not enabled, the early return will allow the program to do the image shift sooner.
In the text box in this group, enter the number of frames in the returned image: 0 for none, a specific number of frames, or -1 for all the frames.
If frame alignment in the SerialEMCCD plugin is selected in the Record parameters, this alignment will be done only for shots without an early return; i.e., the deferred aligned sum computed when using alignment in the plugin with 'EarlyReturnNextShot' is not done here.
Extra delay after image shift
A standard amount of settling time is imposed after each image shift; the extra delay entered here will be added to this settling time.
Adjust beam tilt & astig to compensate for image shift
This option is enabled if you have calibrated the beam tilt needed for coma-free alignment as a function of image shift with Coma vs. Image Shift in the Calibration - Focus & Tuning submenu. If so, then beam tilt will be adjusted appropriately for each Record position, and restored at the end. As of SerialEM 3.8, the dependence of astigmatism on image shift is also calibrated and will be compensated, unless the property 'SkipAstigAdjustmentForIS' is set. In addition, the image shift present when starting the routine will also be compensated for, unless the script command 'AdjustBeamTiltforIS' was run or beam tilt was adjusted by any of the image-shift setting commands that provide the adjustment. The conditions under which the calibration was taken are shown on the following line. For the adjustment to work accurately, the current beam intensity at least should be close to what it was when the calibration was done.
Calibrate Coma vs. IS
Pressing this button will open the Coma vs. Image Shift Calibration dialog and allow you to calibrate the beam tilt adjustments needed to compensate for image shift. Note that the maximum image shift needed for a regular pattern of holes is shown in the summary line below the spinners that select the size of the pattern.
Test Multishot Positions
Pressing this button will start a test of the beam tilt adjustment needed at various positions, either with or without adjustment for image shitf depending on whether Adjust beam tilt & astig to compensate for image shift is checked. See details in Testing Image Location and Coma Adjustment below.below.
Diameter of circles to draw
If the multi-shot circles are being displayed, the diameter of the circles is set to the entered value in microns, unless the option described next is selected to use the illuminated area on a Thermo/FEI Titan. Press tab or Enter to have the image redrawn after entering a new diameter.
Use Record illuminated area for circles in Low Dose
This option appears on a Thermo/FEI Titan and allows the multi-shot circle display to take its diameter from the illuminated area setting of the Record area when Low Dose mode is on instead of from the value entered in this dialog. The display will be updated dynamically as this illuminated area setting is changed. This update works even while this dialog is option, provided that the scope is in the Record area and 'Continuous update' is check in the Low Dose Control panel before opening the dialog, and illuminated area is changed with microscope controls.
For this option to be valuable rather than misleading, the illuminated area readout must be fairly accurate in the range of interest, which may not be the case.
Testing Image Location and Coma Adjustment
Once positions are set up in this dialog, the script command 'TestNextMultiShot 1' followed by 'MultipleRecords' can be used to test whether image positions are suitable. 'TestNextMultiShot 2' or the Test Multishot Positions button can be used to measure the amount of beam tilt adjustment needed at various positions, either with or without an adjustment for image shift, depending on the state of the Adjust beam tilt & astig to compensate for image shift checkbox. The details of this operation are as follows: