Montage Setup Dialog
This dialog box allows you to set the parameters for a montage, see the total area to be acquired, and view the parameters after montaging has been initiated. When this dialog is invoked by the Navigator to set up a montage that fits a defined area, it operates in a special mode, referred to as area-fitting mode. In this mode, you can adjust the number of frames and overlap between frames by changing the magnification or a percentage overlap factor. When one of these settings (or the binning or the selection of stage versus image shift) is changed, the dialog will maintain the fit to the selected area by having the Navigator recompute the number and size of frames and their overlap. Montages will be taken with the Record camera parameters by default, but this dialog includes an option to take a montage with the separate Mont-map parameters.
Montages can also be taken in Low Dose mode, with either the Record, View or Search area imaging parameters. Whether a montage file can be acquired into in Low Dose depends solely on whether Low Dose was on when the montage was first set up. Thus, you need to turn on Low Dose mode before setting up a montage to be taken in Low Dose. Several parameters cannot be changed when in Low Dose mode.
Electronic image shift is used in several different ways depending on the options selected below: basic montaging with image shift, hybrid montaging with image shift in blocks and stage shift to the blocks, and realigning to a piece with image shift. If you are doing extensive acquisition in any of these ways, you should see if your microscope is already using the new defaults for image shift settling introduced in SerialEM 4.0 and, if not, whether it is possible to do so. See the descriptions for the ImageShiftDelays and ISdelayScaleFactor properties, and if necessary see the procedures described for Set IS Delay Factor in the Calibrate menu.
Montage Size-Related Controls
Camera radio buttons
If you have multiple cameras, each available camera will be listed here so that you can specify or see what camera will be used for montaging. The current camera will be the one initially selected. If you select a different camera, it will become the current camera when a montage is started. The camera selection is fixed in Low Dose mode.
When not in Low Dose mode, this setting is initially based on the current microscope magnification, but you can change the magnification if desired using the spin buttons. SerialEM will automatically switch to this magnification whenever acquiring a montage. You can also change the magnification after montaging has been started, but you will have to confirm this change the first time you do so. In area-fitting mode, changing magnification will change the number of frames and may change their size and overlap. In Low Dose mode, the magnification is simply that of the Record or View area and cannot be changed here.
This setting is initially based on the current value in the Record parameter set (or possibly the View set in Low Dose mode), but you can change the binning when setting up the montage. If you do so, the program will adjust the exposure time by the square of the change in binning, but you will also be warned to adjust the exposure and drift settling parameters yourself. If you have already started montaging, you may be able to change the binning if it is compatible with the size of the pieces. In area-fitting mode, changing the binning will cause the fit to be recomputed.
This value is based upon the magnification, the binning, and the calibrations in SerialEM.
Number of pieces in X and Y
You can set the number of frames of the montage in the X and Y directions by entering numbers in the text boxes or using the spin buttons. In area-fitting mode, changes here will be superseded if you change any of the settings that cause the fit to be recomputed.
Piece size in X and Y
You can set the size of each piece in X and Y by entering values in these text boxes. The default is the full size of the CCD camera. In area-fitting mode, changes here will be superseded if you change any of the settings that cause the fit to be recomputed.
Overlap in X and Y
You can set the amount of overlap between pieces by entering values in these text boxes. The default is 10% of the frame size of the CCD camera when doing a montage with image shift. When using stage movements, the overlaps are constrained by a minimum percentage and by a minimum absolute distance in microns. You can use the Reset button to restore the values to the 10% default for image shift or the minimum required for stage montages. These values may change when new piece sizes are entered.
In area-fitting mode, these fields are disabled and overlap is adjusted with the minimum overlap controls instead.
Minimum overlap factor (Montage with stage movement only)
These controls allow you to adjust the minimum amount of overlap required when doing a stage montage. At lower magnifications, a percentage is enough to guarantee enough overlap, but an absolute minimum amount of overlap is needed to get enough overlap at higher magnifications, because of the imprecision of stage movement. Use the spin buttons to adjust the minimum overlap between pieces as a percentage of the largest piece dimension, and enter a value in the text box to change the minimum overlap in microns. Overlaps will be increased to meet an increased minimum, but will not be decreased when you decrease a minimum until you press the Reset button.
In area-fitting mode, the overlap will change automatically not matter which direction the minimum is changed, and the number of frames may change to maintain the fit to the desired area.
Total area and Update
The total area is computed based on the pixel size, number of pieces, piece size and overlaps. It is recomputed whenever you push a button or leave a text box, but to be sure the value is current, just push the Update button.
Options Controlling How the Montage is Acquired
Move stage instead of shifting Image
Select this option to have the montage acquired by moving the stage instead of using electronic image shift. Image shift is quite accurate but has a limited range at medium and high magnifications (generally less than ~15 microns, but less than ~3 microns on some microscopes such as 300 KV Tecnai/Polara). Stage shift can be used when these limits need to be exceeded. Note that in LM mode, image shift has a much higher range, so you may be able to use image shift instead of stage shift at the highest LM mode magnifications. In area-fitting mode, the fit will be recomputed and it may not be possible to change this setting.
Image shift in blocks with IS up to xx microns
With this option, a hybrid montage can be done with a mixture of stage and image shift. Image shift is used within the biggest area for which the image shift at the corners of the block does not exceed the entered maximum value, and stage shift is used to move to the center of each block. A separate entry is stored for montages in and out of low magnification mode. The value should be smaller than the maximum range of image shift allowed by the microscope in the respective mode. If the value exceeds the maximum image shift allowed when fitting to a polygon, the program will show '***' after 'microns' and warn you about this disparity when the dialog is closed. These maximum shifts are determined by property settings and larger shifts may be feasible for your situation.
When non-square pieces are being acquired, the number of image shift positions may be different in X and Y if that will result in a more nearly square area for the block. The overlap between pieces is the same regardless of which kind of shift is used, so the overlap must be set as large as it would need to be for a montage using just stage shift. If the montage size is not larger than the block size, the program falls back to doing a regular image shift montage. This option will save time only if the image shift settling times are fairly short (see paragraph on settling times at the beginning of this page). Before adopting this method for routine use, you should assess whether it is indeed faster than a regular stage montage.
This option radically changes the order of piece acquisition. Without blocks, pieces are acquired in columns (along the Y axis of the camera). With blocks, a whole block is acquired (in columns just like a montage of that size), then the next block is acquired in Y, etc. After finishing a column of blocks, the program shifts over by the block size in X to start the next column.
Focusing in blocks can be used with this option, but the focus blocks are constrained to be the same as the image shift blocks.
Skip pieces outside Navigator item
With this option, it is possible to define a polygon in the Navigator and have each montage that is taken include only pieces that have some portion inside the polygon. This option is not needed when taking a montage that was fit to a polygon but would be useful when taking multiple montages in a region (e.g., supermontaging). After checking the option, enter the position of the desired polygon item in the Navigator table (numbered from 1), or enter 0 to use the current Navigator item or the polygon that was used to define the supermontage including the current item. In other words, the option is only needed when supermontaging, and the value just needs to be 0. The legality of the item number will not be checked until the montage is started. If the Navigator is not open at that time, this setting is ignored.
Do full rectangle; ignore list of pieces to skip
When a montage is set up by the Navigator, it will compose a list of pieces that do not need to be acquired because they fall outside the polygon used to define the montage area. The option allows one to acquire the complete array of pieces, including ones on the list to skip.
Make map from each montage if Navigator open
If this option is checked, then when you take a montage manually, the program will make it a Navigator map. It is available regardless of whether Navigator is open, in case you open it after setting up the montage. It is turned on when you set up a montage through the Navigator; otherwise it is off by default.
Close file when montage is done
If this option is checked, then when you take a montage manually, the program will close the file after the montage is done. In addition, if the file is used to acquire montages with the Navigator and is already open when Acquire at Items is started, it will be closed when the Navigator acquisition finishes.
Use Montage mapping, not Record parameters
If this option is checked and enabled, montages will be taken with the Mont-map camera parameter set instead of Record parameters. The option is most likely to be useful for taking maps, which often involve different exposure time and binning from montages for data acquisition; but it could also be useful if you routinely take montages for other uses with different parameters from the Record ones. The option is disabled if one of the two options described next is selected in Low Dose mode, or if the Mont-map parameters are being hidden with the No Mont. Map Params option in the Camera menu.
Use View parameters in Low Dose mode
If this option is checked, montages in Low Dose mode will be taken with the View camera and imaging parameters instead of Record parameters. This is useful for mapping in Low Dose because a View shift offset can be set to align the View and Record areas (see Shift offset for View or Search). When this option is turned on, the program may turn the option for moving the stage on or off depending on the field of view of View images. When using View parameters is turned off again, it will restore the previous setting of the stage move option if that is known. In Low Dose mode, if neither Record nor Search is defined with a magnification in imaging mode, this option will be checked and disabled, because it is the only way that montages can be taken.
Use Search parameters in Low Dose mode
If this option is checked, montages in Low Dose mode will be taken with the Search imaging parameters instead of Record or View parameters. The Search camera parameters will be used, unless the Use View for Search option is selected in the Camera menu. Search mode may be useful for low-magnification mapping in Low Dose because a Search shift offset can be set to align the Search area with higher magnification areas. When this option is turned on, the program may turn the option for moving the stage on or off depending on the field of view of Search images. When using Search parameters is turned off again, it will restore the previous setting of the stage move option if that is known.
Acquire with Multiple Record routine
If this option is checked, montages in Low Dose mode will be acquired with the Multiple Record routine. There are several consequences:
Use continuous mode with settling factor
This option is enabled for cameras where true continuous acquisition is available: Gatan, DE, and some Tietz cameras. It allows a montage to be acquired by selecting from the stream of images taken in continuous mode. It is most effective for OneView and Rio cameras, where drift correction can be used in continuous mode to produce good-quality images even if there is some residual drift in the images. It could also be useful for taking good-quality montages with other cameras if the normal settling time after movement is used, or for taking maps with a reduced settling time. The settling factor is a multiplier applied to the normal settling time that is allowed to elapse after an image or stage shift before starting a regular image acquisition. In continuous mode, the program will allow the modified settling time to elapse before asking for the next image that comes from the camera. You will need to experiment with this factor to determine what value gives adequate quality for your application. Small values like 0.25 to 0.5, or even 0, may be acceptable with a OneView doing drift correction. Separately from this, there may be drift in the first image (or loss of frames with a OneView or Rio camera when the drift correction is used). This can be handled with a drift setlling value for the particular camera parameter set being used, which will be converted to a number of exposures to drop before accepting the first image after continuous mode is turned on.
Turn off Drift Correction for stage montage
This option is enabled when a montage with stage movement is to be taken with a OneView camera. If it is turned on, the Drift Correction setting can be left on in the Record parameters but the correction will not be used when taking images for the montage. The reason this may be desirable is that the OneView discards frames that it fails to align, giving images with counts that are lower proportional to the number of lost frames. This can easily happen when taking maps of areas that contain some empty regions. The program keeps track of two settings of this option, one for regular and one for "high-quality" stage montages (described in the next section). The initial defaults are to have the option off for high-quality montages and on for regular ones.
High-Quality Stage Montage Options
Use settings for high-quality stage montage
Use this option to activate a collection of settings that are relevant only when doing large stage montages for acquisition of data to be analyzed, as opposed to maps. It is not enabled in Low Dose mode. These options appear when the box is checked:
Autofocus at each piece
Select this option to have the autofocus routine run before acquiring each piece. This will have no effect in Low Mag mode. Turn this on will turn off 'Autofocus in blocks', and vice versa.
Autofocus in blocks of pieces
Select this option to acquire the montage in square blocks of a size selectable with the spin button, and to have the autofocus routine run at the center of each block before starting the block. This option changes the order of piece acquisition, as described above for the Image shift in blocks option. These two options can be used together, but are constrained to have the same blocks for image shift and focus. In fact, the spinners for the two options change the same underlying number, with the number of pieces in each dimension shown for this option and the size in microns shown for the Image shift in blocks option. When both options are selected, the spin button here is not enabled and the block size shown here is determined by the maximum image shift value for blocks.
Repeat until drift is below a limit
Select this option to have autofocus repeated if the drift, in nanometers per second, is above the limit that you enter in the text box. The option is available both when autofocusing at each piece and when using autofocus together with image shift in blocks, but is not available when just focusing in blocks. The program will repeat the autofocus up to five times if necessary, then go on to acquire the piece. You must have the Drift Protection option in the focus menu selected for this operation to work.
Realign with image shift up to a limit
This option can be used to deal with several problems with stage montages: irregular movements that produce variable amounts of overlap; long-term drift that results in overlaps between very long columns being inadequate because a long interval passes between successive passes up the column; and even some inaccuracy in the stage calibration. For every piece after the first one, the program picks an adjacent, already-acquired piece to align to, moves the stage to the target location of the piece to be acquired (based on the actual position at which the neighbor was acquired), autofocuses if that option is selected, then sets image shift in order to take an image that overlaps as much as possible with the one from the adjacent piece. The amount to shift is limited by the number in the text box, in microns. The image is aligned and image shift is then set to take a picture that is in exactly the right location relative to the neighbor. However, if the alignment shift is larger than the percentage indicated in the text box for Maximum alignment shift as % of piece, then it will be ignored.
This option cannot be used together with either focusing in blocks or image shifting in blocks. Using image shift here can be slow because of the settling time required after an image shift; see the note at the beginning of this page. I
With this option selected, the program will remind you to calibrate image shift for the current magnification, but only if the possible error in overlap caused by plausible errors in the current shift calibration is large enough. Large amounts of image shift as a fraction of the frame size, and narrow overlap zones, will trigger this reminder.
Go from center out; anchor at lower mag
When using realignment with image shift, this option can be used to protect against the possibility of poor correlation alignments at the periphery of the area being acquired (e.g., due to grid bars or empty resin). Pieces will be acquired from the center outward: for each column, the program starts in the middle of the column and goes down then up; columns are done from the center leftward, then from the center rightward. In order to find the center again for the second half, an 'anchor' image will be taken at the magnification selected with the spinner and stored in a temporary file before the montage is started. The program will also use this image to estimate the backlash occurring between the two directions of movement. For reliable correlations, the image needs to have a field of view at least twice as big as the possible drift in stage position over the first half of the acquisition. Initially, the program will try to pick a magnification that gives a field of view of at least 20 microns as long as that is not in low mag; thereafter it will remember your setting. Be sure that images can be taken at the chosen magnification without the aperture occupying more than the corners of the image.
Delay time after moving stage
The value here sets the delay time between stage movement and the acquisition of an image. This delay may need to be longer than the default to give images without drift. Currently, the default of 2 seconds (or whatever is set in the properties file with 'StageMoveDelay') will be applied as a minimum delay and a value here of less than the default is ignored.
Skip correlations used to align pieces
The correlations that are usually used to assess how well pieces align can consume a significant fraction of the acquisition time, especially when the option is checked in the Montage Control Panel to treat the montage as 'Very sloppy'. Use this option to disable the correlations. Note that correlations will not be done for montages with more than 512 pieces, regardless of this setting.