Tilt Series Extra Output Dialog

This dialog is used to control the saving of images to additional image buffers or files during a tilt series. The final Record or montage overview can be binned down and saved in buffers shown in a separate window. Four different types of images can be saved to files. First, the various tracking images taken by the Tilt Series Controller, and autofocus pictures, can be saved to files. Second, additional Record images can be saved at different defocus levels, or at different energy filter settings. These extra Record images are taken after the normal Record or montage is taken, and are saved to a separate file from the main tilt series to maintain the linkage between tilt and image number in the latter file. An entry will appear in the log for each image saved to an extra output file.  Third, in Low Dose mode a pair of Trial images can be saved, one from the other side of the Record area.  These images are taken like extra Record images, after the normal Record is taken, but they are not constrained to being at the same binning as regular Trial images.  Fourth, in STEM mode, images from other detectors than the primary one can be saved into a file.  When there is more than one extra Record image, they can each be saved into a separate file instead of all into the same file, and named automatically with suffixies entered here.

When setting up tilt series for acquisition in the Navigator, the current values in this dialog are incorporated into the tilt series parameters only for the first item designated as a tilt series.  If you then open this dialog from the Tilt Series menu and make changes, those changes will be overridden by stored parameters when you press TS Params in the Navigator to adjust parameters for any items.  To change extra output settings, press TS Params for the item where you want the changes to take effect, then press Set Up Extra Output at the bottom of the Tilt Series Setup dialog to open this dialog and make the changes.

Stack Record/Overview images in separate window

Select this check box to display binned down Record or montage overview images in a window separate from the main window. This window will initially be opened at half the size of the binned images, and its title will be the name of the tilt series output file. When the window is the active one, hot keys can be used as usual to scroll through and zoom the display. (It may be necessary to click inside the window with the mouse to make it active.) Images will be labeled by their number in the buffer stack and by the Z value of the corresponding image in the output file.

This option can be turned on and off at any time during a series. If the window is closed, a new window will be opened when the next image is ready to be shown, but the previous images will be gone. At the end of tilt series, the window will no longer accept new images but it will be left open until you close it. If you start a new tilt series, a new window will be opened for its images.

Bin to N KB/image

This spin button lets you control how much the images are binned by adjusting the maximum memory allowed for each image. The memory requirement is the primary concern when keeping a stack that may contain over 100 images; for this reason, the displayed images are converted to bytes as well as binned down. The kilobytes per image and the maximum size of a square image are shown as you change this setting. The maximum size is limited by default to 512 by 512 pixels; this limit can be changed with a 'MaxStackWindowSizeXY' entry in the properties file.

View, Focus, Trial, Preview check boxes

These check boxes govern whether images of the particular type will be saved during the tilt series. You can change these selections at any point in the series.

File # and Name spin buttons

The spin buttons allow you to select a file number in which each kind of image will be saved. They can be set to file numbers up to the maximum number of image files allowed. If there is already an open file at a file number, its name will be shown; otherwise the selector will show 'Not open yet'. When the tilt series is started or resumed, the program will check the validity of the indicated file for each of the kinds of images being saved. Basically, each file must be compatible with images of the given type, based on the current camera parameters for taking that type of image. You can save different kinds of images into the same file as long as they have the same dimensions. You may not save any extra images into the main tilt series output file.

Montaged files cannot be selected for saving anything except extra Record images in TEM mode (i.e., a montaged file is not allowed for saving opposite Trials or STEM mode).  You will most likely want a montage that is the same size as the main output, but this is not required or done automatically by the program; you have to set up the montage of the needed size when you open the output file.

If only one type of extra output is selected, and you enter the suffixes described next, the file number here is irrelevant. 

Suffixes for extra files to open in Nav. Acquire

This entry sets one or more suffixes so that the Acquire at Items routine will open the corresponding number of extra files whenever it opens a regular file for output. Enter the suffixes separated by spaces (a suffix cannot contain a space). The name of each extra file will be the root of the regular filename, plus the suffix, followed by the extension of the regular filename.  The extra files will be opened before the regular file.  They will be closed when the regular file is closed (at the end of acquisition or before opening a new file).  If any file has not had images written to it, it will be removed after being closed, so you can use this command to specify the maximum number of files needed, even if some items do not require all the files.  The entries here will be overridden by the 'SuffixForExtraFile' script command if that is run prior to acquiring a particular item.  If only one type of extra output is selected (e.g, just extra Records), the program will automatically set the starting file number to the first file opened, and the file number here is irrelevant.  If there is more than one type of output selected (or if you use 'SuffixForExtraFile' prior to SerialEM 4.1, 23-Oct-22), you need to anticipate how many other files will be open when starting the batch acquisition and set the file numbers here appropriately.  Specifically, if no other files are open, you need to set the lowest file number to 1.

Save multiple Records in consecutive files

Select this option to have each extra Record image at one tilt angle saved into a separate file, where the file numbers are consecutive from the one selected with the spin button.  With the option turned on, a line below this will show whether these additional files are open yet.

Extra Record/Trial radio buttons and text box

These buttons select whether one or more Record images are saved at different focus or filter settings or from different STEM detectors, or whether a pair of Trial images are taken. When a focus or filter series is selected, the text box is enabled and a series of entries can be made. Entries must be separated by spaces.

For a focus series, the values to enter are the change in focus from the current defocus target. Separate multiple values with spaces, e.g., '-2 -4 2'.

For a filter series, each entry consists of two values separated by a comma. The first value specifies the slit width: 0 to filter with the current width, -1 to take an image with the slit open, or a value greater than 0 to filter with the given width. The second value specifies the change in energy loss from the current value; positive and negative values are allowed. For example, if the regular Record image is set up to be zero-loss filtered with a slit width of 20, then entries of '-1,0 0,40 50,20' would result in three extra Record images: an unfiltered image, one with the regular slit width of 20 eV and an energy loss of 40 eV, and one with a slit width of 50 eV and an energy loss of 20 eV.

When you select the option to save images from multiple STEM detectors, the dialog will show a line listing the numbers of the detectors that will be acquired simultaneously with the main Record image, based on the current selection in the Record parameter set.  Up to 6 channels can be acquired from simultaneously during a tilt series; this limit could easily be raised.  Separate acquisitions are needed to acquire from more than this number of detectors, or to acquire from ones that cannot be used simultaneously with the main Record detector.  Enter a space-separated list of additional channels to acquire in separate acquisitions in the text box.  Note that when acquiring from more than 3 simultaneous channels during a tilt series, the number of rolling buffers is set to the number of simultaneous channels, and the alignment and read buffers are shifted to higher buffers.

Changes for Extra Records/Trials

The options in this group allow you to change the beam or the camera acquisition parameters for the extra Record or Trial images. Redundant methods are provided because there may be problems with one or two of them in any given case.

• Enter a number different from 1 in 'Change intensity by factor of' to have the intensity changed by that factor and restored at the end.
• Select the 'Change Spot size to' checkbox and set the spinner to the desired value to have the spot size changed.
• Select the 'Set exposure to' checkbox to have the exposure time and drift settling set to the values in the text boxes. The drift settling will be applied only for if appropriate for the given camera and shuttering mode, even though the 'Drift settling' text box is not disabled in such cases.  The exposure time entry can be one or more values separated by spaces, where a 0 means to leave the exposure unchanged from the value for the previous image, and there do not need to be as many values as extra Records.  For example, if you have a filter series with 5 extra Records, you can enter '0. 3. 0. 7.' and the exposure will be the same as a regular Record for the first extra Record, 3. for the second and third, and 7. for the fourth and fifth.
• Finally, if you are doing a pair of Trial images, you can select 'Set Trial binning to' and set the binning for these images with the spinner.

Keep file with bidirectional anchor images

Normally, the program stores the initial anchor image for a bidirectional tilt series in a file and deletes the file at the end of the tilt series.  If this option is selected, the image used to realign to the anchor is appended to the file and the file is not deleted.

Make low-tilt map if run from Nav Acquire

Use this option to have a map entry created in the Navigator table for each tilt series run from the Navigator Acquire at Items routine, making it possible to browse through low tilt images for all the series afterwards.  For bidirectional or dose-symmetric series, the first image will be used; for unidirectional series, an image at or near zero-tilt will be used.  Montaged and single-frame tilt series are treated differently: for the latter, the image will be saved to a separate file, with an option to filter it first.  The file will be named with the root of the tilt series file and '_LTmap.mrc', and the map item label will be based on the label of the item currently being acquired plus '-LT'.  For a montage, the map entry will point to that view in the tilt series file itself, and filtering cannot be done.  That map entry is adjusted after a bidirectional or dose-symmetric series is reordered.

Filter non-mont: cutoff ... falloff

Select this option to apply a high-frequency Fourier filter to the low-tilt image from a single-frame tilt series before saving it as a map.  The cutoff entry is the start of the Gaussian falloff, referred to as 'radius2' in other places, and the falloff entry is the sigma value for the Gaussian, referred to as 'sigma2'.  Both are in reciprocal pixels that range from 0 to 0.5.