Navigator menu commands

The Navigator menu has commands for opening the Navigator window, operating on Navigator files, and performing less common Navigator operations.

Setup Corner Montage command (Navigator - Montaging & Grids sub-menu)

Use this command to set up a montage to capture the area within the current set of corner points. The following description applies to this command and next two that involve fitting a montage to an area.  Outside of Low Dose, be sure to go to the desired magnification before activating this command.  In Low Dose mode, the program will use the pre-existing settings of the 'Use View in Low Dose Mode' and 'Use Search in Low Dose mode' options to determine how to fit the area.  If it finds that the fit requires too many pieces, it will switch to a mode with a larger field of view.  More likely, it will open anyway with the wrong area selected and you will have to change this selection.

 You will be opening a new image file for the montage, so if there is already a file open the program will ask whether you want to close it. The program will compute the number of montage frames in X and Y required to capture the area, and decide whether the montage should be acquired with image shift or stage movements. If the area of the Record parameter set is substantially smaller than the camera field, you will be asked whether you want the frame size to be kept smaller than the current Record area. You might want this if the beam is too small to fill the whole camera field. Otherwise, the program will make the frames as large as possible. Unless there are too many frames, you will then go on to the Montage Setup dialog box, in which you will see the number of frames and their size and overlap. If the size of the montage is not reasonable (e.g., bigger than 20K by 20K pixels), outside of Low Dose you can change the magnification to adjust the number of frames and achieve the desired image size.  In Low Dose mode, you can only switch to using View or Search mode instead. You can also adjust the percentage overlap, which may also change the number of frames needed to fit the desired area. Once you have set up a reasonable montage, go on to the File Properties dialog box then the Save As dialog box.

After the montage is defined, the program will compose a list of pieces that are not needed to capture the intended area. These pieces will be excluded when the montage is acquired.

Setup Polygon Montage command (Navigator - Montaging & Grids sub-menu)

If the current Navigator item is a polygon, use this command to set up a montage to capture the area within the polygon. It works the same as the Setup Corner Montage command .  Once the montage is set up to capture an area of a particular shape, it can be used to capture the same area at other locations without drawing a new polygon.

Setup Full Montage command (Navigator - Montaging & Grids sub-menu)

Use this command to set up a montage to capture the whole grid area. A set of points will be composed that contain the grid area. The command then works the same as the Setup Corner Montage command, except that in Low Dose mode, it will automatically switch to using the current Low Dose area if it is View or Search and  has a pixel size 5 times larger than the other area.  After the montage is acquired and displayed, if the Navigator is open, you will likely see a dashed red rectangle indicating the limits of stage movement.  Note that if there are image shift offset calibrations, the coordinates of the red rectangle are based on the offset-adjusted stage coordinates that the Navigator uses.  This means that you may not be able to get to all positions within the rectangle at the low magnification of the grid map, but you will be able to get to them at a magnification closer to the one that is the basis for the offset adjustment.

Set Grid Limits command (Navigator - Montaging & Grids sub-menu)

Use this command to enter lower and upper limits in X and Y for the full grid montage.  Specifically, these limits will be used to construct the polygon that is fit when setting up the full montage, but not to limit the stage positions visited to acquire a montage covering that polygon.  Actual stage movement can be limited only by the 'StageLimits' property in the SerialEMproperties.txt file, and this property, not the setting here, determines the coordinates of the dashed red rectangle that will be drawn on images that extend beyond the limits when the Navigator is open.

A dialog box will come up for each limit; enter the desired limit in microns or 0 to use the default limit that is shown, which is the property-based limit that can be modified with 'StageLimits'.  If you enter a number bigger than one of the defaults, you may get farther out to the corners of the usable area, but the stage will still not be moved beyond the property-based stage limits.  If you want access to the full range of motion on a JEOL scope, you need to enter or change the 'StageLimits' property to be '-1190 1190 -1190 1190' (or less extreme values if appropriate).

Multiple Grid Operations command (Navigator - Montaging & Grids sub-menu)

Use this command to open the Multiple Grid Operations dialog and control acquisition on multiple grids.  If the property 'ScopeHasAutoloader' is set to 0, this menu item appears as Multiple Operations on Grid and the dialog is modified to work appropriately when doing the same operations on a single grid.

Reopen Multi-grid Session command (Navigator - Montaging & Grids sub-menu)

Read in a file saved by the Multiple Grid Operations dialog or multi-grid routine, open the dialog if it is not open yet, and load the contents into the grid table in that dialog.  Session files are named with the format 'multiGrid_date_#.adoc'.  The file chooser will open on the last session file saved, if any.  After a file is read in this way, the session information will be saved into a file with the same name.

Clear Multi-grid Session command (Navigator - Montaging & Grids sub-menu)

Use this command to clear out the grid table in the Multiple Grid Operations dialog and re-initialize the session.

Identify Grid on Stage command (Navigator - Montaging & Grids sub-menu)

Use this command to specify which grid is actually on the stage, in case the program has lost track of this information.

Load All Grid Maps command (Navigator - Montaging & Grids sub-menu)

Use this command to load all grid maps that have been acquired in a session with the Multiple Grid Operations dialog, or to load all apparent grid maps from the currently open Navigator file when there is either no multiple grid session or no grid maps available in a session.  The command is thus useful for a different style of working, where multiple grid maps are all in one file at different registrations.  In this case, the program looks for the lowest magnification at which maps were obtained, in LM only, and loads as many maps taken at that magnification as possible.  In either case, it will load the maps in buffers past the read buffer if possible, and thus ordinarily past the rolling buffers, but they may start in lower buffers if necessary to fit all the maps. If this does not fit your needs, the script command 'LoadAllGridMaps' allows you to specify the starting buffer to load into.  Once all these maps are loaded, it is tempting to add points to any one of them, but this would often result in points in the wrong Navigator file.  As long as the Multiple Grid Operations dialog is open, the program will prevent adding points to a map that is not in the table for the open Navigator file.  When you try to do so, it will print a warning up to 5 times in the log.

Find Holes in Regular Array command (Navigator - Montaging & Grids sub-menu)

Use this command to open the Hole Finder dialog, find circular holes in a support film, and make Navigator points from them. The Navigator must be open to open this dialog.

Add Grid of Points command (Navigator - Montaging & Grids sub-menu)

Use this command to add a set of points on a regular rectangular lattice. Four different kinds of grids can be defined: points in a rectangle; points on a defined rectangular lattice to fill a polygon or map; points along a line, and points at a defined spacing to fill a polygon or map. For the first two kinds, the position of the lattice is specified by a group of 5 points, three at corners of a rectangle and two more adjacent to one of the corners. Here, holes that should be marked with a point are shown with 'X' and the rest of the holes are shown with 'o'.

X      o      o      o      o

o      o      o      o      o

o      o      o      o      o

o      o      o      o      o

X      o      o      o      o

X      X      o      o      X

To make all of the points be in one group, press the 'Add points' button and mark all 5 points before pressing 'Stop Adding'. As long as one of the points is the current item, you can select this command. The program will then ask for the label of a polygon that you want to fill. If you leave the text box blank, it will create points for all positions in the rectangle indicated by the corners. If you enter a valid label for a polygon that contains the points (specifically, the centroid of the points), then it will add points to fill the polygon, extending the rectangle beyond the corners that you have marked if necessary.  If you are filling an elongated region, you can reduce errors from extrapolation by marking the corner with two adjacent points near the middle of the region instead of near one end.

For a large grid, it may be difficult to mark the two adjacent points accurately enough to end up with the right number of points between them and the endpoint.  You can overcome this with a group of 7 points, where the points along one axis would look like

X      X      o      o      X      o      o      o      o      o      o      o      o      o      o      o      o      o      o      o      X

and similarly along the other axis.  The additional point between the adjacent pair and the endpoint (X) allows it to get a more accurate initial estimate of the interval between points, so that when it divides the longer distance by this interval and rounds to the nearest integer, it comes out with the right number. Placing this point at the square root of the total number of points along the line should be optimal (e.g, at the fifth point if there are 25).  (The additional point needs to be far enough out to give a sufficiently accurate initial estimate but not so far that it mis-estimates the number of points along the line to that point.)  If you start with a 5-point group and the placement fails, you can add the two points to the same group by turning on Edit mode, selecting any point in the group with the left mouse button, and adding the two intermediate points with the middle mouse button.

Once you have added a grid with a 5- or 7-point pattern like this, you can add a grid in a new area with the same spacing and orientation by simply adding a single point in the new area.  It must be the only point in the group, so press 'Add points', add the point, and press 'Stop Adding'.  If you are not filling a polygon, the point should be at the same corner of the region to fill as the corner of the 5-point pattern with two adjacent points.  If you are filling a polygon, the program will test for whether either this point itself, or the center of the grid that would extend from that point as its corner, is in the polygon.  Thus, you can put the point near the middle of the polygon to reduce extrapolation errors.  (The test for the point itself being in the polygon was added in SerialEM 3.7.4).

To add a line of points, simply add a group of three points to indicate the extent and spacing of the line, such as:

X    X    o    o    o    o    o    o    X

As long as one of these points is the current item, the command will be available and will add a line of points between the endpoints.

Finally, to add equally spaced points within a polygon or map but not on a defined lattice, simply make the polygon or map be the current item and select this command. Then enter the desired spacing in microns.  Alternatively, keep one of the points in the group as the current item and start this command.  You will be asked to identify a polygon or map to fill with the grid, either by the item's label or by the negative of the item's index in the table. 

When adding points to fill a polygon or the rectangle of a map, the points will not be placed right up against the border of the polygon. Rather, a square box around each point is required to be fully within the polygon. The size of this box as a fraction of the spacing between points is controlled by the GridInPolygonBoxFraction property, whose default value is 0.75.

Grid points can be added in two different patterns. One is a zigzag path, in which points go in alternating directions between successive rows or columns. This was originally the only pattern in SerialEM and was designed to minimize stage movement when moving between points. The other pattern goes in the same direction in each row and column, and proceeds in an overall direction away from the location of the Focus area in Low Dose mode. This pattern ensures that focusing is done in areas that have already been acquired from.  However, this pattern is not used when added points are placed into separate groups.

Add Grid Like Last One command (Navigator - Montaging & Grids sub-menu)

After adding a grid of points with the previous command, you can use this command to add another grid of the same kind without having to answer any queries.  'Of the same kind' means that if the last grid was added on a group of points or a single point, the command can be used if a point is selected; whereas if the last grid was added with a polygon or map selected, a polygon or map must again be selected.   If you added a grid with a group of points or single point selected and chose to fill a polygon or map with the grid, then the program will search for the smallest polygon or map containing either the point itself or the center of the pattern extending from the current point as its corner and fill that area.  In other words, when you use a single point to reuse a previous 5 or 7 point pattern, you can put the point right in the middle (as of SerialEM 3.7.4) or you can put it in the corner of the grid square, even outside the polygon, as long as the position at the center of the previous pattern, starting at that point as its corner, is inside the polygon.

In sum, this command should work just the way you want: add a point at the corner of a new area to be filled and run the command, which you can do with the menu accelerator Alt-N-M-L.  The easiest way to add a single point is to place the marker there first, then press 'Add Marker'.

Divide Points into Groups command (Navigator - Montaging & Grids sub-menu)

When this option is selected, the set of points added with the Add Grid of Points command will be divided into multiple groups.  The middle point of the group will be the first point, and its distance to every other point in the group will be less than the maximum radius set with the Set Group Radius command.  With this arrangement, you can use the option in the Acquire at Items dialog to focus only on the first point in a group, and other points will be within a certain distance of the location where focus was last done.

When points are divided into groups, the program will display the result, tell you the number of groups and points per group, and ask if you want to keep this grouping.  If groups are too large or small, just press No and all the points will be removed.

Set Group Radius command (Navigator - Montaging & Grids sub-menu)

Use this command to set the maximum radius used for dividing points into groups as described above.  Sometimes groups will be smaller than they could be for a particular radius, because the total extent in each direction of the grid gets divided into groups as evenly as possible.

Set Multi-shot Parameters command (Navigator - Montaging & Grids sub-menu)

Use this command to open the Multiple Record Setup dialog and set parameters for using image shift to take multiple Record images in a circular pattern around the current position, on a regular array, or in a custom pattern of positions.

Set Hole Order for Regular Array command (Navigator - Montaging & Grids sub-menu)

Use this command to set the order in which holes in a regular array are acquired.  The choices are: 1) Jump from the center to the corner then do alternating rows in opposite direction, referred to as a zigzag path, so as to minimize the shifts between holes after the initial jump.  2) Jump to the corner and do all rows in the same direction, a raster pattern.  3) Start in the center and do concentric squares, following a spiral path to minimize all shifts between holes.  If the array is not square, the spiral path is followed in a the largest possible square, then the extra rows or columns are added in a zigzag path that still minimizes shifts.

Combine Points for Multi-shots command (Navigator - Montaging & Grids sub-menu)

Use this command to open the Multiple Hole Combiner dialog, which lets you convert a regular array of points marked for acquisition into a subset that are set up for multiple Record acquisition.

Show Shots when Show Acquire command (Navigator - Montaging & Grids sub-menu)

This command toggles whether selecting the 'Show Acquire area' checkbox in the Navigator window will also result in a pattern of circles being drawn based on the parameters set in the Multiple Record Setup dialog when that dialog is not open.  The circles will represent the area covered by the beam, assuming the beam size specified there.  The pattern will be drawn around the marker point if one is present; otherwise it will be drawn around the current item, if it is marked for acquisition, or around multiple selected items.  However, it will not be drawn if the current item is set to acquire an incomplete pattern (i.e., in which some positions are being skipped), so that it is clear which positions will be acquired for that item.  If Edit mode is not on, use Shift-Left-click to turn off the marker and see the pattern around the current point.  If Edit mode is on and there are multiple hole positions in a regular (non-custom) pattern, then Shift-double-Left-click near one of the positions will remove it from the pattern.

Show Whole Area for All Points (Navigator - Montaging & Grids sub-menu)

Use this command to have the envelope of all acquire areas from multiple Records drawn around all points marked for acquisition, when the 'Show Acquire area' checkbox is turned on in the Navigator window.  If the multi-hole pattern around a point has been modified by deleting positions, then boxes representing the Record area will be drawn instead of the envelope.

Autocontour Grid Squares command (Navigator - Montaging & Grids sub-menu)

Use this command to open the Autocontouring Grid Squares dialog, which can make contours around grid squares at an intensity threshold, allow you to set criteria for eliminating some contours, and convert selected contours into Navigator polygons.

Reverse Contour Colors command (Navigator - Montaging & Grids sub-menu)

Use this command to invert the order in which colors are assigned to groups of contours generated by the Autocontouring Grid Squares dialog in inverted order, from magenta and purple to orange and red, instead of from red through magenta.  This could be useful if you use only 5 or 6 groups and find the colors on the blue-magenta end more distinguishable.

Keep Colors for Polygons command (Navigator - Montaging & Grids sub-menu)

This command toggles whether the groups of contours generated by the Autocontouring Grid Squares dialog are assigned the same colors when they are converted to polygons, rather than given the default polygon color.

Setup Super Montage command (Navigator - Montaging & Grids sub-menu)

Use this command to add a set of rectangles at the positions of overlapping montages, laid out on a rectangular grid that is aligned to the camera axes. First, you must have montaging set up with the desired number of pieces and magnification. Specify a center position in the active image by clicking with the left mouse button. Then select this command. You will be asked to enter the number of montages in X and in Y then the number of pixels of overlap in X and Y. A set of polygons will be created, numbered by their positions in the super montage. To try again with different parameters, just use the Delete button in the Navigator to delete the whole group of items.

To get fairly accurate overlaps between montages, use the following procedure. First take a montage that encompasses the whole needed area, at a lower magnification if necessary. On a Thermo/FEI microscope, it is recommended that you use the option to realign with image shift (see Acquiring High-Quality Montages of Large Areas in Montaging and the last paragraph of Returning to a Position Reliably in Using the Navigator for details). Turn on 'Align pieces in Overview', and also have 'Sloppy montage' on as well if this is needed to get the pieces properly aligned. Make this montage be a map. Then go to the working magnification for the supermontage and set up the montage file. After you answer the queries about number of montages and overlaps, you will be asked if you want the positions to be laid out at regularly spaced image positions rather than stage positions. Do not select this option unless the pieces are indeed properly aligned in the area covered by the supermontage (there can be errors in alignment elsewhere). Also, only select this option if you are going to use the 'Realign to Item' routine at some point to get to individual montage positions. One approach would be to check 'Acquire' for each of the new montage position items and then run the Acquire at Items command with the option checked to 'Realign at item'. This will yield montages that can be blended and analyzed for overlap.

The Setup Super Montage command was originally called Aligned Super Montage, and there was also a Skewed Super Montage command to lay out montages on a grid that is aligned to the specimen axes. Such montages require more overlap to avoid gaps between montages, and the stitching programs in IMOD work well with the aligned supermontages, so the skew capability has no apparent merit.

Polygon Super Montage command (Navigator - Montaging & Grids sub-menu)

Use this command to add a set of rectangles at the positions of overlapping montages that will fill a polygon. First, you must have montaging set up with the desired number of pieces and magnification, and the current Navigator item must be a polygon. After you select this command, specify whether the positions should be marked for acquisition. The supermontage will include any montage that is needed to cover the area within the polygon, with a little allowance for errors in positioning. You can then use the 'Skip pieces outside Navigator item' option in the Montage Setup dialog box, to have each individual montage include only the frames inside the polygon. If you use an entry of 0 there to specify the current Navigator item, be sure to reselect the polygon item after defining the supermontage.

Add Circle Polygon command (Navigator - Montaging & Grids sub-menu)

Use this command to add a polygon that is a circle. First mark the desired center point by clicking in an image. Then select this command and enter the desired radius in microns.

Polygon from Corners command (Navigator - Montaging & Grids sub-menu)

Use this command to make a polygon from the current set of points marked as corners.  After the polygon is made, you will be asked whether you want to delete all of the corner points. If you answer No, they will be retained but no longer be marked as corners.  With this command, you can use corner points to define one polygon after another, and then set up each polygon for automatic acquisition into a different montage file.

Open command (Navigator menu)

Use this command to open the Navigator window.

Read & Open command (Navigator menu)

Use this command to read in a Navigator file as well as open the window if it is not already open. The file will become the current file for saving into.

When a Navigator file is read, each map file will be located if possible.  The program will look first at the path specified in the Navigator file.  If this fails, it will next look in the same directory as the Navigator file.  Next it will test whether the path starts with './'. '.\', '../', or '..\', and if so, then the program will look for the map file at this path relative to the location of the Navigator file. (SerialEM saves an absolute path for the map file, but other software might use relative paths.)  Finally, it will use the information in the Navigator file about where it was last saved to see if there was a relative path from the Navigator file to the map file previously, and if so, it will look at this relative path from the current location of the file.

If any map file cannot be found, the program will issue a message listing such files.   If each such map  file is moved into the directory of the current Navigator file, its maps will be found when needed.

Merge File command (Navigator menu)

Use this command to read in the items from another Navigator file and merge them in with the items currently present in the table.  Duplicate items in the file being merged will be eliminated, with no change in the properties of the existing items.  If an item has a label that matches that of an existing item, it will be changed by adding '-1' to it (or '-2' if the label with '-1' on it exists, etc.).

The file being merged in need not be in the same directory as the current Navigator file.  Map files in the merged file will be located as described in the previous command, except that files not at their defined paths will be sought at locations relative to the file being merged in, not the current Navigator file.

Save command (Navigator menu)

Use this command to save Navigator items to a file. The file is a simple tab-delimited text file with the properties of each item. If there is no current Navigator file, you will enter the Save As dialog box to specify the file, which will have the default extension .nav. When you select this command again, the entries will be saved to this file.

Save As command (Navigator menu)

Use this command to save Navigator items to a file different from the current file. You will enter the Save As dialog box to specify the file. The new file will become the current file for saving into.

Close command (Navigator menu)

Use this command to close the Navigator window. If the contents of the window have changed since it was last saved, you will be asked if you want to save them to a file.

Set Point Label Threshold command (Navigator - Options submenu)

Use this command to set a threshold for skipping the drawing of most labels on points when they belong to groups bigger than the threshold.  Enter the maximum group size for which all labels should appear, or 0 to draw labels regardless of group size.  The labels of the first and last points in the group will be drawn in groups bigger than the threshold. Also, the labels of up to five points around the currently selected point will still be drawn even if the selected point is in a group larger than the threshold.  Labels will also be drawn for points whose distance from the previous point is greater than 40 microns.

Use Item Labels in Filenames command (Navigator - Options submenu)

Toggle this menu item on to incorporate the item label in the filename created when a new file is to be started for an item.  If there are no filenames defined for previous items when you select New file at item or Tilt series, the filename will be initialized to the item's label plus the appropriate file type extension, and that name will appear in the file chooser window that comes up for defining the filename. You can add more text or replace the label as you desire for this file.  If you add text that might be the same as any label (i.e., having numbers), be sure to add it before the label.  If there is a previous filename that is the basis for a new name and it still contains the label of its respective item, then the current item's label will be substituted for the previous one.  The last occurrence of the label will be replaced if it occurs more than once.  To stop having names based on the label, either turn off this option or remove the label from the next name that you define.  To start having names based on the label when there is a preceding filename without its label, press Set Filename and insert the current item's label into the name (but again, after any other numbers in the name).

This option should be particularly useful for generating names that correspond between the two axes of dual-axis tilt series.

Autosave Nav File command (Navigator - Options submenu)

Toggle this menu item on to have the Navigator items saved to a file periodically if they have changed. If there is no currently open file, the program will save to a file named AUTOSAVEmmddhhmm.nav, where 'mmddhhmm' are two-digit values for month, day, hour, and minute. This file will be removed when you save to a file or when the program exits (if you elect not to save at that time). However, if the program crashes, you should find the file in the directory in which your SerialEM shortcut starts the program.

Write as XML command (Navigator - Options submenu)

This command toggles an option to save the Navigator file as an XML file instead of in autodoc format.  Either one can be read back in by SerialEM.  The autodoc writing is significantly faster because it is optimized to write each line directly to file, whereas to write an the XML file the program builds an autodoc data structure, builds an XML structure from that, then writes out the XML.  This will matter only for very large numbers of points, over 10,000.

Convert Maps to Bytes command (Navigator - Options submenu)

This command toggles an option to keep maps in memory as bytes. The option does not affect how maps are stored in the image file (typically as integers). Byte conversion will reduce memory usage by 2/3, so that the memory required is just one byte per pixel. The intensity scaling determined by the Black and White levels in the Image Display control panel will be associated with an image when a new map is created. This scaling is then applied to convert the image to bytes, both when the map is defined and when the map is reloaded from file. This scaling could make it impossible to see some light or dark image details. There are two ways to prevent such scaling problems. First, adjust the Black and White levels to avoid truncating image intensities before designating an image as a map. Second, use this menu entry to turn off the conversion option, load the map, adjust the Black and White levels as desired, and turn the option back on.  This option is not saved in the settings file between sessions; it can be turned off by default with the general property 'ConvertMapsToBytesDefault 0'.

Load Maps Unbinned command (Navigator - Options submenu)

This command toggles an option to have montage maps always loaded unbinned, i.e., with overview binning of 1. This is almost always what one wants, but if you are short on memory and do not need to see a map at full resolution, you can turn off the option and set a higher overview binning in the Montage Control panel . However, if the map file is open, you must make it the current open file before setting the binning in the control panel.

Use Current LD Params in Realign command (Navigator - Options submenu)

This command toggles an option to use the current Low Dose parameters for Realign to Item instead of the Low Dose parameters set when the map was acquired.  The option applies only when staying in Low Dose mode for the Realign operation, which occurs as long as the current camera, magnification, and probe mode (on a Thermo/FEI scope) or alpha (on a JEOL) match the map condition.  By default, the Realign operation imposes the map conditions when realigning to a map.  This option is available because there are situations where it is advantageous to take the map under different conditions.  For example, more dose might be desired for the map to allow either better visualization of features or more reliable montage piece alignment, while less dose can be used for correlating with those maps.

Try Scaling in Realign (Navigator - Options submenu)

This command toggles an option to try to adjust for specimen shrinkage when doing Realign to Item .  On the first round of the realignment, where an image is aligned to the center of a map frame, the program will search for the image scaling that makes it align best to the map frame.  This scaling is then used to adjust the target position (which is not at the center) for the shrinkage that occurs.  The scaling will not be done if the program is in Low Dose mode prior to the Realign operation.

Search + and - Scaling in Realign command (Navigator - Options submenu)

This command toggles an option to try scalings both below and above 1 when searching for the best scaling in Realign to Item.  This might be useful when aligning to a synthesized map or one made on a different microscope with somewhat lower magnification.

Run Script in Multi-shot (Navigator - Options submenu)

This command toggles an option to run a script in place of the Record acquisition in the Multiple Records routine.  The Multiple Records cannot itself be run from script, it must be run from Navigator Acquire at Items.  There are probably other restrictions of what the script can do, which will be revealed by malfunctions and crashes.  If you want a Record to occur, you must include the 'Record' command in the script.

Set Script to Run (Navigator - Options submenu)

Use this command to select the script to run instead of taking a Record in the Multiple Records routine.  When you select this command, a dialog will open with a drop-down list in which you can select the script by its name or number.  It is best to have a (short) name assigned to the script so that if scripts are rearranged, or a different set is loaded, the program will not run the wrong script.

Transform Items command (Navigator menu)

Use this command to transform items from one or more other registrations to the current registration. The program will find a transformation whose complexity depends on the number of registration points available. Just a shift will be found with 1 point, a rotation and shift with 2 points, rotation, scaling, and shift with 3 or 4 points, or a full linear transformation including stretching and possible image inversion with 5 points. All items except the registration points will then be transformed and associated with the current registration. The program will give a message about how well the points fit the transformation equation. If the fit is very poor, you can undo the transformation with the command described next, fix the problem, and redo this command.

If stretch in the stage coordinate system has been calibrated with the Stage Stretch command , then transformations based on 2 - 4 points will incorporate this stretch, and the report on the transformation in the log will include the text '(corrected for stage stretch)'. When transforming after a large specimen rotation, such transformations may not be very accurate in the absence of such a calibration, but the calibration allows reliable transformations with only two or three points.

Undo Transformation command (Navigator menu)

Use this command to undo the last transformation done with the Transform Points command . Each item will be returned to its former registration, even if items were transformed from more than one registration. This is the only way to undo a transformation of imported items.

Change Registration command (Navigator menu)

Use this command to change the registration of an existing item without changing the current registration. This is different from changing the registration point number, and in fact the command cannot be applied to registration points. If you need to change the registration of a registration point, turn off its the 'Registration point' button before changing the registration.

Align with Rotation command (Navigator menu)

This command opens the Align with Rotation dialog box , which allows you to align a current image with a map taken prior to rotating the specimen. You can also use this alignment to transform items into the new registration, although this transformation is of limited accuracy at long distances from the point being aligned. In order to open this dialog, the map must be in the buffer that images are read into (i.e., just load the map), and there must be either an image in buffer A, or a montage overview in buffer B and montage center in A, at a different registration from that of the map. Thus, after rotating the specimen, increase the current registration in the Navigator, then take an image at the same magnification as the map.

Shift to Marker command (Navigator menu)

This command provides a potentially simple method of shifting items without having to make registration points. One way to use it is to add a single point at a recognizable feature on an existing map. Then take a new image, click on the feature with the left mouse button, and select this command. The Shift to Marker dialog will open with information about the shift and some choices for applying to a subset of items and for saving the shift for reuse. If you press OK, all items at the registration of the image that was clicked on (typically the current registration) will be shifted by the same amount. (See the dialog help for explanations of the options there.) Another way to use this command is to run the Realign to Item command on a map or an item located in a map. When the procedure is done, take an image, click in the center of it, and select this command. Items will be shifted by the error in stage position found in the procedure. Finally, the command can be used to shift registration points to fit imported items. Suppose you import a map, work out registration points, transform the map, and then import another map that is shifted in position. Then select one of the registration points, click on the corresponding point on the new imported map, and select this command. The registration points will be shifted but the map will not, so the points should appear at the right place in this map. This map can then be transformed into registration with EM maps.

This menu item is enabled either when the conditions are satisfied for doing the Shift to Marker operation, or when there are saved shifts. In the latter case, the dialog will open to allow shifts to be inspected or removed, but none of the other controls will be available.

Apply Saved Shift command (Navigator menu)

Use this command to shift a set of items at a particular magnification for which a shift was saved in the Shift to Marker dialog. Specifically, if a shift was saved for a magnification, maps taken at that magnification that have not been shifted with the Shift to Marker procedure can be shifted, as well as any points or polygons marked on them. First, you will be asked to confirm or specify the magnification of the stored shift to use. If there has been more than one shift saved for that magnification, you will next need to specify which one to used, identified by the magnification of the image on which the marker point was placed. Finally, you be asked to confirm the specific amount of shift to apply. See the Shift to Marker dialog help for some scenarios in which this is useful.

Undo Last Shift command (Navigator menu)

Use this command to undo the shift imposed by the 'Shift to Marker' or 'Apply Saved Shift' commands. It is disabled if the 'Transform Points' command is used.

Import Map command (Navigator menu)

Use this command to define an image obtained elsewhere, such as on a light microscope, as a map. First, a dialog will open to allow you to select the file. You can select TIFF and DigitalMicrograph files (dm3 or dm4 type) as well as MRC files, and you can use color images. Then the Navigator Import dialog box will open to allow you to specify the section number, a stage position, a registration number, and a transformation between stage and image coordinates. If you import a TIFF file, the stage position and the stage to image transformation may be automatically selected if there are tags in the file defined in the SerialEMproperties.txt file. The dialog will also allow you to make a color overlay of the selected file and the currently selected Navigator item, if the latter is an imported map of the same size. Imported maps are originally forced to have a registration number separate from those of non-imported items, so points marked on imported maps will not appear on other images, and vice-versa. However, once you transform the imported map into the same registration as the other images, then points marked on the imported map will appear on other images and can be used to move the stage to a desired location.

When points are marked on a map, the program stores the ID of the map that they were drawn on.  This "drawn on" ID is used to help decide what map to use for the Realign to Item routine, but an imported map cannot be used in that routine.  When the imported map is transformed into registration with another map, the "drawn on" ID of any non-registration points marked on the imported map will be switched to the ID of the map that was registered to, and any points added after the transformation will also be given that "drawn on" ID.  Thus, that map will be preferred for realigning to.  In order for this to occur, all the registration points must be picked on just the two maps, one imported one and one native one being aligned to.

Rotate Map command (Navigator menu)

Use this command to rotate the map in the active buffer into the correct orientation for the current magnification and registration, or to rotate an imported map by a desired angle and optionally flip it. Specifically, the rotation may include two components: the relative rotation between the magnification at which the map was acquired and the current magnification; and whatever rotation and mirroring around the X axis resulted from any transformations that have been applied to the map item. Points will still display correctly on the rotated map, and new positions can be marked on it. A map can be rotated more than once, or it can be reloaded and rotated to a new orientation.

If a map is imported and has not yet been transformed into registration with native EM maps, then the program will ask you the angle by which to rotate the map (counterclockwise is positive), then ask whether to flip the image around the X axis after rotation.

Adjust for Backlash command (Navigator menu)

With this command, the program will measure the stage backlash at the location of the current item and then adjust the item's stage position and record the backlash direction used.  When stage backlash is substantial, this can improve the accuracy of returning to that location.  The measurement involves dropping magnification if necessary, or going to the View area in Low Dose mode, taking an image, then moving away and back by the backlash amount, taking a new image, and aligning it.  The stage position of the item will be adjusted by the alignment shift.  The backlash used is the same as the backlash for a montage at the starting magnification.  The command is available only for an item with a directly measured stage position (i.e., a map, or point created with Add Stage Pos), and only if the stage is still at the original position.

Note that whenever a map has a backlash recorded for it, the program will correct for backlash in the corresponding way when moving to a point marked on the map.  Montages made with stage movement automatically have a backlash recorded, but other maps may require this backlash adjustment, unless they were taken at a location where the stage was in a known state with respect to backlash.

Backlash Settings command (Navigator menu)

Use this command to open a dialog box for settings that control whether the backlash adjustment routine described just above will be run when a new single-frame map is added.  The dialog has a text box for specifying the minimum size of map for which the program should even consider running the routine.  There is no maximum size setting, but the routine will not be run for a map taken in low magnification. There are three choices:

1. Do not run the adjustment routine automatically or ask whether it should be run.
2. Ask whether the adjustment routine should be run when making a new map larger than the minimum size.
3. Run the adjustment routine automatically when making a new map larger than the minimum size.

When you use the Anchor Map function, the program will automatically adjust for backlash, if necessary, unless you have chosen option 1.  The higher magnification item that you are anchoring will also have its position adjusted, provided that the stage is still at the same location.

Open State Dialog command (Navigator menu)

Use this command to open the Imaging State dialog box , which can be used to store and set different states for image acquisition. Because the ability to manipulate microscope and imaging state is particularly helpful when using the Navigator, this dialog will open automatically along with the Navigator if it was open the last time that the Navigator was open. If you close the dialog before the Navigator, however, you need to use this command to reopen it. You can also use this command to open the dialog without opening the Navigator.

Set Map Acquire State command (Navigator menu)

Use this command to return to the conditions under which the currently selected map item was acquired, to facilitate acquiring another map in the same state. The microscope state includes magnification, intensity, spot size, and energy filter settings. The camera acquisition state includes the binning, exposure time, and drift settling of the Record parameter set. Low dose will be turned off if it is on. If the file containing the selected map is still open in SerialEM, it will be made the current file. If the file is not open, it will be reopened so that a new map can be saved into it. This command performs the same operation as the Set Map Acquire State button in the Imaging State dialog box.

Restore State command (Navigator menu)

This command will restore the microscope and camera acquisition state that was present before the last time that 'Set Map Acquire State' was run, or before the imaging state was set from the Imaging State dialog box. If a file was opened when 'set Map Acquire State' was run, that file will be closed again. If the file that was current at that time is still open, it will be made the current file again. Low dose mode will be restored to its state prior to the state changes. This command does the same thing as the Restore Prior State button in the Imaging State dialog box.

Acquire at Items command (Navigator menu)

Use this command to acquire images or maps or run a script or tilt series at a series of items marked for acquisition with the 'Acquire' or 'Tilt series' check box in the Navigator window. There must be an image file open before the command can be used to acquire images or maps, unless 'New file at item' has been set for the first item to be acquired. If montaging is to be used, this means that the montaging must be set up already. Be sure to have magnification, intensity, and exposure times set before starting the acquisition, unless an imaging state has been set for the first item. This command opens the Acquire at Items dialog box, which allows various options to be set.

End Acquire command (Navigator menu)

After starting an 'Acquire at Items', use this command to stop the Navigator from going on to the next item after finishing the current one.  If the acquisition is already paused after finishing an item, the command will stop acquisition immediately.

List Files/Series/States command (Navigator menu)

Use this command to get a list in the Log window of everything that has been scheduled to happen in the course of running an 'Acquire at Items' operation. Specifically, the list will show the files that will be opened and their properties, the imaging states to be set when files are opened, and the tilt series that are set up to run.  For a Low Dose state (marked as LD), the list will include (after 'F pos') the focus position along the axis between low-dose areas, the rotation of the axis if any, and the offset from center of a Focus subarea in binned pixels. In addition, the Focus position will be listed for any items that have it stored, regardless of whether a file is to be opened or a state set.

Realign to Item command (Navigator menu)

This command can be used to return accurately to the position of an item by correlating with available map images. The item must be located within, or must itself be, a map whose extent is large enough to allow the position to be found reliably. Specifically, the map must be at least 3 microns in extent (or more, if the property file specifies a larger limit). Maps taken in LM mode will not be used for these correlations unless they are from a relatively high magnification in LM where images can be taken with the objective aperture in.  The limit for how low a magnification it will use is controlled by the property RealignItemMaxLMField. There are basically four different situations in which this procedure can be used:

1. The item is not a map, just a point or polygon. It must be located within a big enough map, but it can be very near the edge of the map. In this case, the program will first move the stage to the center of the map, or to the center of one frame of the map if it is a montage, then take an image and align it to determine the error in stage position. It will then move the stage to the position of the item, take a new image, and align it to work out the error from that small stage movement. If this procedure has been run on a nearby area recently enough, the program is able to use the results of previously aligning to the map center and go directly to the second round of alignment at the item position.
2. The item is a map that is not located in a larger map, so it must itself be large enough for reliable localization. The program proceeds as just described, except that it ends up aligned to the center of the map.
3. The item is a map located within a significantly larger map. In this case, the larger map must be the minimum required size, but the item being aligned to can be small. The program proceeds as above to find the item position by correlating with the larger map. Then it does a third round of alignment by raising the magnification to that of the item itself, takes a new picture, and aligns it with the map item using image shift. This image shift should be relatively small provided that the position was found accurately at the lower magnification, and that the calibration of image shift offsets between magnifications is still accurate.
4. The item is a position on a map at a lower magnification than that at which images are to be acquired, and the operation is run in a way that allows it to do a third round of alignment between an image at the higher magnification an appropriately scaled and rotated portion of the map centered on that point.  This method is only available when running through the Navigator Acquire at Items procedure or from a script command, not through the menu or the button in the Navigator window.  If the position can be marked accurately enough on the map, it has three advantages:
   • There is no need to store a map at the higher magnification as in situation 3.
   • The scaled-up reference image can be bigger than the camera frame, which allows shifts larger than half the field of view to be found reliably, provided that the image is not dominated by periodic structures.  This is also an advantage over the virtual maps of Schorb et al., Nature Methods 16: 471-477 (2019).
   • If the higher magnification image has a large enough field of view (10 microns by default) and the routine is able to skip the first round of alignment, it will skip the second round as well, go directly to the item position, and just do the third round.  This is particularly useful for aligning at an intermediate magnification to an LM map, as it avoids going in and out of LM.
   This method requires a camera parameter set or Low Dose area to be designated for the image to be aligned.  In Low Dose mode, the area must be at a higher magnification than the map.  Outside of Low Dose, the routine must start at a higher magnification than the map.  The third round will simply be skipped if these conditions are not met.  Parameters for this method are set in the Navigator Align Setup dialog.

When a map is defined, the program stores the brightness, spot size, and exposure settings used to take the map images, as well as the state of the energy filter if appropriate, and these settings are used in this routine to take the images for correlation. The beam size and centering must be robust enough to work at the different magnifications. In other words, if the beam shifts between the different magnifications, it must be spread enough at one of the magnifications so that it still covers the field without being centered.

The previous state of the microscope is restored after the procedure when it is run with this command from the menu or the corresponding button in the Navigator window. When it is run from a script, the state may or may not be restored depending on what is specified in the script command.  However, when the routine is called while in Low Dose mode, the state is always restored by going back to the previous Low Dose area after taking images in the same conditions as the map.

When in Low Dose mode and realigning to a map taken in Low Dose, the routine will attempt to remain in Low Dose if the current parameters of the area with which the map was taken match the parameters used for taking the map.  The match is not considered good enough if there is a change in the camera, the magnification, or the probe mode (on Thermo/FEI) or alpha (on JEOL); or if the defocus offset (for a View or Search map) has changed by more than a limit set by the RealignItemFocusChangeLimit property, which is not set by default.  If the match is not good enough, the program will internally leave Low Dose mode by going to the Record area first, which can waste many seconds.   If the match is good enough, the routine will substitute the map parameters into the Low Dose area and take images with those parameters, unless an option is set in the Tasks-Specials Options submenu to realign with the current area parameters.

In most cases, the routine will adjust for misalignments between magnifications even if you have not selected the 'Adjust image shift between mags' option in the Alignment & Focus control panel. Specifically, in situations 1) and 2) above, if the microscope state is being restored after the routine runs, then the feature being aligned to should appear centered at the restored magnification. In situation 3, the feature will be centered at the magnification of the map item being aligned to.

Setup Align/Realign (Navigator menu)  

Use this command to open the Navigator Align Setup dialog and set parameters and set up a template for the Align to Template command.  Some options for resetting image shift after such an alignment can also be set; these parameters are also used when Navigator Acquire at Items runs the Realign to Item routine.

Force Center Align (Navigator menu)  

This command makes the Realign to Item routine forget previous information about a recent alignment to the center of the map and do the first round of alignment to the map center.  By default, Realign to Item will skip that first round if the same map was aligned to recently and the alignment error was low enough.  If the first round fails, it may be necessary to use this command to prevent the skipping on the next attempt.

Erase Periodic Peaks in LD (Navigator menu)  

This command toggles whether alignment correlations for Realign to Item in Low Dose mode are done after trying to identify and erase periodic peaks in the FFTs used for the correlations.  Erasing of peaks seems to work well for gold grids and is essential when working with ones having hexagonally spaced holes, which are much closer together.  It does not work reliably for regular carbon grids, which may not have enough remaining non-periodic signal.  This option alone determines whether erasing is done in Low Dose mode; the general option in the Image Alignment & Focus control panel determines whether erasing is done outside of Low Dose mode.