Process menu commands

The Process menu offers the following commands:

Min/Max/Mean Computes min, max, mean and S.D. of active image or subarea.
Zero-tilt mean Computes mean of area foreshortened by tilting.
FFT Computes full-sized Fourier Transform of image in active window
Binned FFT Computes FFT of image binned to 1024 or binned by 2-4.
Live FFT Computes FFT of images taken in continuous acquisition mode.
Side by Side Shows FFT in a window side-by-side with main window.
Automatic Single FFTs Takes FFT of new single-frame images automatically if Side-by-side selected
Circle on Live FFT Draws circle(s) on the Live FFT images
Set Defocus for Circles Allows you enter a defocus to have circles drawn at corresponding CTF zeros
Do Ctffind On Click Computes defocus/astigmatism by fitting to Thon rings after clicking near first zero.
Set Ctffind Options Sets options for the Ctffind operation when clicking.
Set Phase Plate Shift Allows you to enter the phase shift from a phase plate
Rotate Left Rotates image in active buffer counterclockwise.
Rotate Right Rotates image in active buffer clockwise.
Crop Image Crops out image inside drawn box or box around drawn line
Reduce Image Reduces image in size by a selected factor with antialiasing
Make Color Overlay Combines 2 or 3 gray-scale images into a single color image
Show Cross-corr Shows cross-correlation from the current autoalignment
Autocorrelation Computes autocorrelation of image in Buffer A
Fix Dark X-rays Removes X-rays from image in Buffer A using dark criteria
Set Dark Criteria Allows you to set criteria for dark X-ray removal
Fix Image X-rays Removes X-rays from image in Buffer A using image criteria
Set Image Criteria Allows you to set criteria for image X-ray removal

In the following, the "active window" means the current buffer in the main window or a single-image window if it is the active window; the stack of images created as extra output from a tilt series is excluded even if it is the active window.

Min/Max/Mean command (Process menu)

Use this command to obtain the minimum, maximum, and mean values of the image in the active window or a subarea of it. The information will appear in a message box, or in the Log window if it is open. The computation is done on the active window, which may not be the main window. If there is a box drawn on this image (which you can do with Ctrl-Shift and the left mouse button), the statistics within the box are given, along with the size and location of the box.  If there is no box drawn, the program will also report the pixel size of the image.

Shortcut Key:CTRL+I

Zero-tilt mean command (Process menu)

Use this command to obtain the mean value of the approximate area in the current buffer that corresponds to the full field at zero tilt. For example, if the tilt is 60 degrees, the magnification is such that the tilt axis is near vertical, and the image is 1024 pixels wide, then the mean will be taken of the central half of the image with X between 512 and 768. Note that the mean is taken of the current buffer in the main window, even if it is not the active window.

Shortcut Key:CTRL+Z

FFT command (Process menu)

Use this command to take a full-sized Fourier transform (FFT) of the image in the active window. The FFT will be placed into buffer A and existing images will be rolled to higher buffers to the extent specified in the Buffer Control Panel, just as when a new image is acquired.

If you click in an FFT with the middle mouse button, the status bar will show the frequency and periodicity (resolution) of the clicked position.

If you have an FFT that shows Thon rings, there are two ways to determine the defocus: either by automatic fitting to several Thon rings with the Ctffind module (see the Do Ctffind On Click command below, which activates this feature), or by manually clicking on the FFT if that feature is not active.  

For manual measurement, click right at the location of the first minimum (the first zero of the CTF) with the left mouse button.  The program will draw a circle through the clicked location and circles at the expected positions of several more minima (5 total by default).  The estimated defocus will be displayed in the middle pane of the status bar.  Once the circles are drawn, you can click again near any one of the circles to refine the fit to the minima in the FFT.  For the inner circles, you have to avoid clicking midway between two circles or it will think you are picking a new first zero position; for the outer two circles it will always refine based on one of those two circles.  Click in one of the corners or very near the middle of the FFT to remove the circles.

The defocus is computed using the voltage determined from the microscope, an objective lens spherical aberration value of 2.5 mm and an amplitude contrast ratio of 0.07.  The property 'FFTZeroParams' can be used to modify these parameters or the number of circles drawn, but this is not essential.  The estimated defocus is quite insensitive to the value of the spherical aberration.  The default aberration was changed from 2.0 to 2.5 in SerialEM 3.7 (8/24/18) to be about midway between the true value for Tecnai/Polara (2.26 mm) and the value of 2.7 for Krios, Talos, and Glacios.  The amplitude contrast ratio affects mostly the position of the first circle, so an estimate refined from higher order circles will also be insensitive to this parameter.

The equations used currently give invalid results for higher-order zeros at a defocus smaller than about -0.3 and for the defocus itself at a defocus smaller than about -0.15, so fewer rings and eventually no rings at all will appear for small defocus values.

Shortcut Key:SHIFT+F

Binned FFT command (Process menu)

Use this command to take a binned FFT of the image in the active window. Before taking the FFT, the image will be padded to be square and binned down by a factor of 2, 3, or 4. The target size after binning is 1024 by 1024; a binning factor of 2 will be used if the image is already this small, and a factor of 4 will be used if the image is larger than 4096 by 4096. The FFT will be placed into buffer A and existing images will be rolled to higher buffers to the extent specified in the Buffer Control Panel, just as when a new image is acquired.

Shortcut Key:SHIFT+B

Live FFT command (Process menu)

Use this command to have FFTs computed automatically when images are taken in continuous acquisition mode. The FFT will replace the acquired image.

Shortcut Key:SHIFT+L

Side by Side command (Process menu)

Use this command to have FFTs displayed in a window next to the main image display window.  This window is a stack of 8 buffers that will roll to save up to 7 FFTs when a new single FFT is taken.  For a live FFT in continuous mode, the top buffer will be continually replaced.  After this option is turned on, the window will be created when an FFT is taken.  You can close the window, and it will be reopened when another FFT is taken.  The window is closed when the option is turned off.

When the FFT window is open, the allocation of the available width between the two windows is based on a fraction that you can adjust and that is stored between uses of the side-by-side display.  Adjusting the right edge of the main window or the left edge of the FFT window will cause both windows to be redrawn with a new fraction.  Adjusting the right edge of the FFT window will change the amount of available width, but only the size of the FFT window will change.  Adjusting the bottom edge of either window will cause both windows to be redrawn with that height.

Shortcut Key: SHIFT+S

Automatic Single FFTs command (Process menu)

Use this command to have FFTs computed automatically for every new single-frame (non-continuous mode) image whenever the side-by-side FFT window is already open.

Circle on Live FFT command (Process menu)

Use this command to have one or more circles drawn on the live FFT images. By default, one circle is drawn, but there are two options for getting more circles.  First, a custom set of circle radii, in reciprocal pixels, can be defined with the FFTCircleRadii property.  Second, the following command can be used to set a defocus for which circles will be drawn at the zeros of the CTF.

Note that if you have left-clicked on a live FFT and there are now circles in the FFT corresponding to a defocus readout in the status bar, you can return to the defined set of circles by clicking near the corner or very near the center of the FFT.

Set Defocus for Circles command (Process menu)

Use this command to set a fixed defocus that defines where circles will be drawn on a live FFT, namely at the zeros of the CTF for that defocus.  Underfocus is negative, as in all defocus readouts.

Do Ctffind on Click command (Process menu)

Use this command to have the Thon rings in an FFT fit with the Ctffind module, adapted from the Ctffind4 program of Rohou and Grigorieff.  There should be at least two zeros (minima) visible in the FFT, preferably more.  Initiate the fitting by clicking near the first zero to inform the program of the approximate defocus (it should work if you click anywhere between the first and second zero, or inside the first zero by up to half the distance between first and second zeros).  The program will solve for defocus and astigmatism, draw ellipses (5 by default) at the zeros, and show the maximum and minimum defocus values in the status bar.  The log will also have the following output:
   defocus 1 & 2: the maximum and minimum defocus values
   angle:  the angle of the maximum defocus (minor axis of ellipse)
   score:  the correlation coefficient between the 2D image and computed CTF

In addition, if the option to compute the extent of good fitting to the Thon rings is turned on with the next command, the log will show the resolution in Angstroms to which the Thon rings can be fit.

Shortcut Key: CTRL+D

Set Ctffind Options command (Process menu)

Use this command to set some parameters for fitting with the Ctffind module.  There will be five entry boxes:

  1. Enter 1 to do the extra computations needed to determine the resolution to which the Thon rings can be fit, or 0 not to.  These computations will increase the fitting time by 10-20%, which may be noticeable.
  2. Enter 1 to draw half-rings on the image out to the resolution determined by these extra computaions if they were done, or 0 not to.
  3. Enter 1 to do a slower 2-D initial exhaustive search instead of the default 1-D search with a rotational averaged spectrum.  This search is needed if the astigmatism is large (for example, if analyzing images with beam-tilt) but should not be needed for ordinary amounts of astigmatism.  The 2-D search is parallelized so its speed will depend heavily on the number of computer cores available.
  4. Enter a limiting value to the maximum resolution to which to fit the amplitude spectrum, in Angstroms.  This setting is unusual in that there is a default that may be set in the SerialEM properties file, and you will continue to use the default (even if it is changed) unless you enter a different value here.  If you do enter a different value, you can return to the default by entering 0.  If there is no property setting, the default is 5 if the voltage on program startup is above 120 KV, which is the same as the default maximum resolution in Ctffind4, and 10 otherwise.  SerialEM will choose a maximum resolution higher than this value when the defocus is high and the minimum resolution is a high number as well.  However, with a non-FEG microscope or other situation where Thon rings may never approach this resolution, a higher value may be needed here or in the properties file.  If you click in an FFT with the middle mouse button, the status bar will show the resolution ('Period') at that frequency.
  5. Enter a limiting value to the minimum resolution in Angstroms for fitting when phase is non-zero.  This limit applies either when a constant phase shift is assumed or when the phase is being found in a fit with the 'Ctffind' script command.  This limit provides a maximum value for the minimum resolution and can be used to exclude the first zero from fitting, since its position may be off due to a gradual cut-on in the phase shift at low frequencies.  However, the minimum resolution may end up being higher than this limit since it is also constrained to be least 1.5 times the maximum resolution used for a particular fit.  Enter 0 for no limit.

All fits are done with a box size of 256 for speed, and because high accuracy at the highest frequencies is not needed.  The 'CtfFind' script command gives more flexibility with regard to the fitting parameters, including the ability to set the box size.

Set Phase Plate Shift command (Process menu)

Use this command to define the phase shift imposed by a phase plate (a positive value in degrees).  This phase shift will be taken into account when circles are drawn on a live FFT for the fixed defocus defined by the previous command, or when a defocus is reported after left-clicking on an FFT.  If a non-zero shift has been entered, the defocus in the status bar will be followed by '(PP)'.

Rotate Left command (Process menu)

Rotate Right command (Process menu)

Use these commands to rotate the image in the active buffer counterclockwise (left) or clockwise (right) by 90 degrees. The rotated image will be placed into buffer A and existing images will be rolled to higher buffers to the extent specified in the Buffer Control Panel, just as when a new image is acquired.

Crop Image command (Process menu)

This command is available if you have drawn either a box or a line in the active image (with Ctrl-Shift-left mouse or Shift-left mouse, respectively). Use the command to crop out the image area inside a drawn box, or inside the box around a drawn line. The cropped image will be placed into buffer A and existing images will be rolled to higher buffers to the extent specified in the Buffer Control Panel, just as when a new image is acquired.

Reduce Image command (Process menu)

Use this command to reduce the size of the image in the active buffer with an antialiasing filter, just as is used when zooming the display down below 1.  Enter the reduction factor in the text box; it must be a factor greater than 1 but is not constrained to an integer value, unlike binning.  The reduced image will be placed into buffer A and existing images will be rolled to higher buffers to the extent specified in the Buffer Control Panel.

Make Color Overlay command (Process menu)

Use this command to overlay two or three images in any buffers into a single color image. A dialog will open, in which you specify which buffers to use for the red, green, and blue components of the image. You do this by entering three letters for the desired buffers, or a 0 if no image is desired for one component. For example, you can enter ABC to take red from the image in A, green from the image in B, and blue from the image in C; AB0 to make a red-green overlay of the images in A and B; ABA to make a magenta - green overlay of the images in A and B; AAB for a yellow-blue overlay; ABB for a red-cyan overlay, etc. The images will be scaled by whatever scaling is currently used to display them and will also be shifted by whatever shifts have been imposed by autoalignment or shifting with the right mouse button. (The latter feature could be made optional if users require it.) The color overlay image will be placed in buffer A and existing images will be rolled to higher buffers to the extent specified in the Buffer Control Panel, just as when a new image is acquired. The color image can be saved to an MRC or TIFF file.

Show Cross-corr command (Process menu)

Use this command to show the cross-correlation used to autoalign the image in buffer A to the image in the autoalign buffer.  The correlation will be placed into Buffer A and existing images will be rolled to higher buffers, just as when a new image is acquired.

Autocorrelation command (Process menu)

Use this command to compute the autocorrelation of the image in buffer A.  The correlation will be placed into Buffer A and existing images will be rolled to higher buffers, just as when a new image is acquired.

Fix Dark X-rays command (Process menu)

Use this command to apply the X-ray removal routine to the image in the active window, using the criteria for removal of X-rays from dark references. The image with X-rays removed will be placed in Buffer A, and existing images will be rolled to higher buffers to the extent specified in the Buffer Control Panel, just as when a new image is acquired.

Set Dark Criteria command (Process menu)

Use this command to set the criteria for dark reference X-ray removal. First you will encounter a dialog box asking for a criterion for the absolute amount above the mean, then another dialog asking for a criterion for the number of standard deviations above the mean, then a final dialog asking whether a pixel must satisfy both criteria or just one of them. These criteria replace the ones specified in the property file and will apply only during this session of the program.

Fix Image X-rays command (Process menu)

Use this command to apply the X-ray removal routine to the image in the active window, using the criteria for removal of X-rays from images. The image with X-rays removed will be placed in Buffer A, and existing images will be rolled to higher buffers to the extent specified in the Buffer Control Panel, just as when a new image is acquired.

Set Image Criteria command (Process menu)

Use this command to set the criteria for X-ray removal from images, which is activated for a particular camera with the ShowRemoveXRaysBox property and for individual camera parameter sets with the Remove X rays checkbox in the Camera Setup dialog box. First you will encounter a dialog box asking for a criterion for the absolute amount above the mean, then another dialog asking for a criterion for the number of standard deviations above the mean, then a final dialog asking whether a pixel must satisfy both criteria or just one of them. These criteria are camera-specific; they are stored in your settings file and will persist between sessions of the program.