Parallel Tilt Series Setup Dialog

This dialog allows you to set parameters, measure specimen angles, and create target areas for a parallel tilt series. Important information on running parallel tilt series can be found in the Parallel Tilt Series section in the Tilt Series help page. Here are some general points about operations with the dialog:

Adding points: Three different procedures will initiate the process of adding Navigator points: (1) defining points to fit a plane and measure angles, (2) adding targets on an area map, and (3) adding points to adjust image shift and generate an adjusting transform. Using the Stop Adding button in the Navigator will have same effect as the Stop Adding button here. A map is needed in all three cases; any map is allowed for fitting to a plane, but for image shift refinement and defining adjusting transforms, the map must be the current area map. You may find it helpful to open a Locator window from the Window menu in order to have a full view of the map alongside a zoomed in view in the main window on which to add targets.

References and IS refinement: The dialog provides some flexibility in how or whether the references for tracking at the targets are made, depending on what works best for your acquisition situation. You can either use a high magnification image saved at each target, or a crop of the area map centered on each target. If the former is selected, maps will be saved at each target during the image shift refinement process. Additionally, you have the option to skip collecting references and tracking altogether. You also have the option to skip refinement, if you believe that the conversion of stage positions to image shifts will be sufficiently reliable. During refinement, a separate window will open displaying a view of the area map zoomed in at the current target, to aid in centering the originally marked target. Changes to the zoom or size of this window will persist until you close the window.

Adjusting transforms: Image shifts derived from positions marked on a medium-magnification often do not apply well at the higher acquisition magnification. This dialog uses the same adjusting transforms that are used when correcting image shift vectors for Multiple Records and that can be defined with the Step To & Adjust IS routine in the Multiple Record Setup dialog. There can be several such transforms, defined from different starting magnifications. The dialog will indicate whether one is available at the mapping magnification. Although it is probably better to define an adjusting transform in Multiple Record Setup on a regular holey specimen, where automatic hole centering can be used, this dialog also provides the ability to create an adjusting transform in case the other procedure is not conveniently available. The transform is defined here by marking points in a map that can be accurately located in a Preview, then adjusting image shift to center each feature in a Preview. A zoomed map overview window will be displayed while adjusting, as is done in image shift refinement.

Image types and cleanup: Preview camera parameters will be used for all images at the acquisition mag, namely, images for refining target positions, for tracking references, and even autofocus images in Low Dose mode. Area maps can be taken with whatever parameters you choose. To avoid clutter, almost all additions to the Navigator will be removed when a target area is finalized (completed). After finalizing, there will only be the area map, the parallel tilt series item, and the target map at the center position (if you chose to save references).

Procedure Overview

This dialog will guide you through a procedure that consists of the following steps in general, which you might follow if starting with a new lamella where you may not know the tilt of the specimen. Bold phrases in this list refer to specific buttons in the dialog with the same or similar name.

1. Run Eucentricity.
2. Save Map of the specimen at tilt of 0 or at the tilt where you suspect the specimen is level (the "leveling tilt").
3. Define Points To Fit Plane: Mark locations on that map where the defocus will be measured.
4. Stop Adding & Fit to measure defocus at each marked point and compute specimen angles ("Leveling tilt" and "X pitch angle").
5. Start New Target Area: The program will tilt to the leveling tilt so that the specimen is flat.
6. Acquire a new image or montage at this tilt and Save Area Map of the desired area where targets will be added.
7. In the Alignment at starting tilt section of the dialog, select a method of obtaining references for tracking at targets.
8. Select whether you want to refine target image shifts or Skip image shift refinement, in which case step 10 is skipped.
9. Add Targets on the area map, starting near the center of the area if possible.
10. Stop Adding & Refine the image shift for each target. The program will realign to the first target in the Navigator, then capture a Preview and pause to allow you to refine the image shift:
    • Right-click and drag to center the target.
    • If necessary to search for the desired location, capture new Preview images.
    • After centering a target, Save Target Map/IS or press the S key to save the image shift and, if applicable, save a tracking reference.
    • You may instead Remove Target.
    • Once the image shift (and target map, if applicable) is saved or the target is removed, the program will shift to the next target, capture a new Preview, and prompt you to repeat this process.
    • Abort Refining to abort the whole refinement process for all targets, keeping the targets.
11. You may freely rearrange or delete refined targets in the Navigator except for the first one, which is the center point and cannot be removed or reordered unless you Clear Saved Shifts and redo the refinement process.
12. Abort Area if you want to delete all the added targets and start a new target area (which can be the existing map).
13. Finalize Target Area to store saved image shifts and all other necessary information in one parallel tilt series item and set up tilt series parameters for the item.
14. Setup Tilt Series if you want to open the Tilt Series Setup dialog again, or on later areas, and make changes to tilt series parameters for the finalized parallel TS item.

Descriptions of Dialog Controls

Magnifications for Mapping and Acquisition

If not in Low Dose mode, you can set the magnifications to be used for the area map and for acquisition. This section is hidden in Low Dose mode, where the Record/Preview parameters define the magnification for acquisition.

Defining Specimen Angles

Save Map

This button will make a map from the current image in buffer A or from a montage overview in buffer B.  For a single image in A, it will save it to the current open file if possible, or else ask you to open a file for saving it into.

Define Points to Fit Plane

The Define Points to Fit Plane routine will measure the defocus at points defined on an area, and then compute a least squares fit with those measurements to determine the geometry of the specimen plane (tilt and X-axis pitch angle). After points are defined and the plane fit procedure starts, it runs automatically and deletes all points at the end. This means all added points are temporary and there is no option to continuously update the sample points used for the fit.  This procedure can be run after starting a new target area, up until the point where you start adding custom targets.  It can also be run after finalizing the targets and before starting the next area, in which case the revised values will be stored in the previously finalized parallel TS item.

1. Click Define Points to Fit Plane and mark at least four points on an area map. These points should span perpendicular directions on the specimen plane.
2. Click Stop Adding and Fit. The program realigns to a point in the center of the marked points and switches to the acquisition magnification.
3. The program image shifts to each point and measures the defocus.
4. The program computes leveling tilt and X pitch angle, and the corresponding edit boxes in the dialog are updated with these new values.
5. All Navigator items added for this routine are automatically deleted when the routine finishes, restoring the Navigator table to its previous state (unless the debug output 'N' is set, in which case items will remain in the Navigator for debugging purposes).

Leveling tilt

The tilt angle at which the specimen will be level, i.e., horizontal in the microscope. When starting a new target area, the tilt will be set to this angle.

X pitch angle

The pitch angle of the specimen relative to the X-axis, i.e., its rotation around the Y axis (counterclockwise positive, looking down the axis).

Defining Area & Targets

Add Custom Targets / Add Regular Pattern

With Add Custom Targets selected, targets are created by marking specific locations on the area map.

With Add Regular Pattern selected, targets are created according to the pattern set in the Multiple Record parameters, by one of two ways.  If the currently selected Navigator item is a point, then the targets will follow the multishot pattern centered at the point. If the item is a polygon, the polygon interior will be populated with targets according to the multishot pattern angle and spacing. No image shift refinement is done on a regular pattern of targets; the targets are generated automatically when finalizing the target area.

Rough Eucentricity

Runs the Rough Eucentricity routine.

Eucentricity by Focus

Runs the Eucentricity by Focus routine.

Start New Target Area

Click this button to go to the specified leveling tilt and start a new target area. Starting a target area and making an area map are required steps before you can add targets to refine/adjust.

Acquire

In Low Dose mode, there are four acquire buttons: Search, View, Preview, and Montage, which capture images with parameters from the specified Low Dose area. Preview is always used to refine target image shifts and create new adjusting transforms.

If not in Low Dose mode, the four buttons are View, Trial, Preview, and Montage. Clicking View, Trial, or Montage will capture images with the mapping magnification set at the top of this dialog. Equivalently, Preview will use the specified acquisition magnification.

Save Area Map / Use Map in Buf for Area Map

If an existing map is loaded into the active (currently displayed) buffer, the button is labeled Use Map in Buf # for Area Map and that map will be assigned as the area map for the parallel tilt series area. Otherwise, the button is labeled Save Area Map and it will be enabled if there is a newly captured single image in buffer A or a montage overview in buffer B.  Pressing will create a new map from the A or B buffer. A single image will be saved to the current open file, if possible, or else you will be prompted to open a new file to save the map.

Adjusting Transform Controls

Apply adjusting transform where appropriate

If this box is checked, the adjusting transform described in the status just line below will be applied while refining target image shifts and/or saving target maps.

Create New Adjusting Transform

The parallel tilt series setup dialog provides controls to create and apply adjusting transforms. Click this button to enter a special state of the dialog which will allow you to create a new adjusting transform. Buttons which are normally used to add, refine, and remove targets will have slightly different behavior; The Stop Adding & Refine / Refine IS button will instead be labeled Stop Adding & Adjust / Adjust IS. When this button is clicked, it will start an adjusting routine that closely mirrors the generic target refinement routine described above.

1. Click Create New Adjusting Transform and mark points on an area map. These should be recognizable features that you can accurately align by adjusting the image shift at the specified acquisition magnification.
2. Click Stop Adding & Adjust / Adjust IS. The program realigns to the first marked point and captures an image at the acquisition magnification.
3. Adjust the image shift to align the original intended target. Click Save Adjustment or press the S key when done adjusting.
4. The program image shifts to the next point and captures an image at the acquisition magnification.
5. Repeat steps 3-4 until you have adjusted all image shifts. You can also add more points after saving a round of image shift adjustments, in which case you will be prompted to immediately adjust their corresponding image shifts.
6. Click Save Transform when you are finished.

Whenever you finish adjusting added points, an adjusting transform is computed and information about the fit is displayed in the log. If you delete a point, the transform is recomputed and the updated results will be shown in the log. The log will display the following:

• The average error from fitting the saved adjustments to a transformation matrixn matrix
• The highest fit error, and the Navigator index and label of the corresponding point.
• The entries in the computed adjusting transform matrix.
• If debug output 'N' is set, the fit error for every point used to compute the adjusting transform.

The general procedure to refine target image shifts is outlined earlier in the overview section, but it can be modified with a variety of options.

The option selected here determines which method will be used to obtain reference images for aligning to targets at the starting tilt. Accordingly, the desired option should be selected before refining targets, because they introduce the following modifications to the image shift refinement routine:

• Take Preview for reference: After image shift is refined, the program saves a map of the Preview image centered on the target.
• Extract reference from area map: Instead of a Preview map, a crop of the area map centered on the point marked on the map will be used for alignment.
• No alignment: The refined image shifts will be applied without any additional alignment.

Note that the latter two options rely on the assumption that the refined image shifts match with target locations marked on the area map. In other words, if you are taking Previews for reference, you can modify target locations during the refinement process, since the Preview maps are saved at the same time as the refined image shifts. If you are using extracts, it is meaningful only to adjust image shifts to center the points marked on the map, and attempting to shift the target location will have no effect.

If checked, image shifts will be obtained by converting stage coordinates of the targets drawn on the area map. This means that, if you are saving Preview maps for references, the refinement procedure will run and automatically save all the target maps without stopping and asking you to refine the image shifts. Alternatively, if you are extracting references from the area map or doing no alignment, you can finalize the target area as soon as targets are added, skipping the refinement procedure entirely.

Click this button to begin adding targets to the area map, or, if already adding, click to stop adding without initiating the refinement routine.

Click this button to begin the refinement routine on added points.

After the routine realigns or image shifts to a target and captures a Preview image, you will be able to freely change the image shift and take more images and this button will be enabled. When you are centered on your desired target, click this button to save the image shift (and possibly also a target map) and continue on to the next target. For convenience, you can press the S key instead of clicking this button.

This button will be enabled after the routine realigns or image shifts to a target and captures a Preview image. Clicking this button will remove the current target. If it is the first target in the group, the routine will stop so you can rearrange or edit the rest of the added targets. Otherwise, it will continue on to the next one.

Clicking this button will consolidate all refined targets into one parallel tilt series item that contains all necessary information about the image shifts and relevant options from the dialog. The first time a parallel tilt series item is created in a given Navigator, you will be prompted to define a tilt series output file and the Tilt Series Parameters dialog will open with relevant parameters imported from this dialog.

Click this button to either clear the whole area and start over, or, if some targets have been refined, just clear the saved image shifts so that refinement can be redone.

Opens the Tilt Series Setup dialog for the finalized parallel tilt series item. This is equivalent to selecting the item in the Navigator and clicking the TS Params button in the Navigator.

This closable section of the dialog contains extra options that affect how beam circles are drawn during target area setup, and how which steps are taken during final acquisition.

Enter the maximum amount that you anticipate tilting from the starting tilt of the tilt series. This entry has two effects. Most visibly, beam circles will be drawn as ellipses, stretched perpendicular to the tilt axis based on the tilt angle specified here. Beam circles/ellipses are drawn at all targets while setting up a target area, and are also drawn when the current Navigator item is a parallel tilt series and the Acquire checkbox is on in the Navigator.  The second effect is that the extreme angles of each tilt series will be set to the leveling tilt plus or minus this angle, if you have the option to do so checked in the Tilt Series Setup dialog.

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 or a beam size computed from a beam size calibration on other scopes. Press tab or Enter to have the image redrawn after entering a new diameter.

This option now appears on all scopes and allows the multi-shot circle display to take its diameter from the intensity setting of the Record area when Low Dose mode is on instead of from the value entered in this dialog. On a Thermo/FEI Titan, the illuminated area is used for the size and a size will always be available. On other scopes, a beam size calibration is used if one exists for the current conditions (namely, on the same side of crossover and for the same probe mode on Thermo/FEI or alpha on a JEOL). This option is permanently disabled if there are no beam size calibrations at all, or temporarily disabled when there is no calibration for the current Record settings.

For this option to be valuable rather than misleading, the illuminated area readout or calculated beam size must be fairly accurate in the range of interest, which may not be the case.

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 targets.

This option is enabled if you have calibrated the beam tilt and astigmatism 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. Astigmatism will also be compensated, unless the property 'SkipAstigAdjustmentForIS' is set. In addition, the image shift present when starting the Multiple Record routine will also be compensated for. 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.

Indicate which CTF type to use, if any, and whether to find astigmatism during final acquisition. See Parallel Tilt Series for more details about this choice.

Enter the maximum shift, as a percentage of the field of view, that will be allowed in alignment to targets.  When alignment finds a shift greater than this maximum, no shift will be applied.

If Extract reference from area map is selected for the alignment at starting tilt, then the following additional options will appear:

• Max rotation: the maximum angle in degrees by which high magnification images at targets will be rotated in order to align to the reference extracted from the area map.
• Max scaling: the maximum scaling, as a percentage, to be applied to high magnification images at targets in order to align to the reference extracted from the area map.

The degree of rotation and scaling that need to be allowed for depends on the accuracy of the pixel size and rotation calibrations that are used to scale and rotate the extracts for alignment.  Larger values may be needed if these are not very accurate, but when values are larger than needed, it increases the risk that the alignment will fail.