Align to
This button will align the image in buffer A to the image in the indicated buffer, using cross-correlation. The buffer holding the image being aligned to is called the autoalign buffer. This buffer is determined as follows:
When not in low dose mode, images will be aligned to the first buffer after the rolling buffers, unless the 'Align to B instead of to...' button in the Buffer Control panel is selected, in which B is the autoalign buffer.
When in low dose mode, there are actually two autoalign buffers: the first for images from the Record area, the second for images from the Trial or Focus area. These are the first and second buffers after the rolling buffers. However, if the 'Align to B instead of to...' button is selected, images of all types will simply be aligned to the image in B.
To Marker
If the marker point is set in buffer A with the left mouse button, this button will set the alignment shift of the image to bring the marker to the center of the field. It is equivalent to dragging the marker to the center with the right mouse button. It works even when the marker point is set outside the image, i.e., in the purple border or in the gray area that results from mouse-shifting an image. The same rules about whether the stage is used instead of image shift apply as for dragging with the mouse: if the option Move stage for big mouse shifts is set, the stage will be moved if the fraction of the field or the absolute distance moved exceeds the respective thresholds for these two measures. Also like right-mouse drag, if the Shift key is held down while pressing this button, the stage will be moved regardless of distance and the setting of that option. The hot key Shift P is equivalent to pressing this button without the Shift key.
Clear
This button will set the alignment shift of the image in the currently displayed buffer to zero. If this is buffer A, then microscope image shift will also be changed by an amount corresponding to the image alignment shift. Thus, this button can be used to undo a bad autoalignment.
Reset Image Shift
This button will reset the microscope image shift to zero and move the stage to compensate for the change in image shift. If the specimen is tilted, defocus will also be changed to compensate for the change in Z height of the feature in the center of the field.
Because of inaccuracies in stage movement, image features can end up 0.5 micron or more from their original positions. This button is useful for a quick reset when you are still moving around on the specimen. If you want to keep a feature of interest centered through the reset, then select the Reset & Realign command in the Tasks menu.
Autofocus
When not in Low Mag mode, this button will run the autofocus routine to measure specimen defocus (based on the amount of image displacement when the beam is tilted) then change the defocus to your chosen target value. In Low Mag mode, it will use the same routine if autofocus is calibrated in Low Mag; otherwise, the button will simply set the focus to the standard focus for the current magnification.
Move stage for big mouse shifts
When this check box is selected, moving the image in Buffer A with the right mouse button will result in the stage being moved by the corresponding amount, provided that the move is bigger than a threshold that can be adjusted with the 'Set Threshold Shift' button. Moves less than the threshold amount will cause the usual image shift instead. If you hold down the Shift key while shifting with the right mouse button, the stage will be moved regardless of the size of the move.
Set Threshold Shift
Use this button to set the thresholds for moving the stage instead of shifting the image with the right mouse button. First you will be asked to enter a relative threshold, expressed as a fraction of the size of the camera field. Then you will enter an absolute threshold in microns.
Correct backlash in stage moves
When this check box is selected, backlash correction will be applied in stage movements initiated with the mouse and when resetting image shift. This should result in more consistent shifts in actual position when there is substantial backlash in the stage, particularly when reversing directions. If the stage is known to be in a backlash corrected state, then the movement will typically be done in two steps so that the final position is approached from the same direction as previously. The two steps are not needed when the movement is already the right direction. If the stage is not in a backlash corrected state, the backlash correction used in montaging at the current magnification will be applied.
Center image shift on tilt axis
When this check box is selected, SerialEM will move the origin for image shift laterally from the optic axis to the tilt axis. Resetting image shift will then set the image shift to a position on the tilt axis, and the IS readout in the Microscope Status panel will be 0.0 even though image shift is nonzero (as can be seen in the microscope interface). With this option enabled, lateral movement and focus changes will be minimized during a tilt series. However, turning the option on or off will misalign the objective aperture on the 300 KV Tecnai or Polara. When the option is selected, the program will move the image shift origin when it is started and reset the origin when it exits.
Adjust image shift between mags
This check box will be enabled if the image shift offsets between magnifications have been calibrated. When it is selected, these offsets will be applied whenever you or the program changes magnification, and features should stay centered despite misalignments between magnifications. This can be particularly useful when going between LM and normal magnifications. Just as for the 'Center Image Shift on Tilt Axis' option, the IS readout in the Microscope Status panel will not reflect these offsets. Using this option may be disadvantageous in situations where image shift is limited by the objective aperture or by intrinsic limitations, since an unknown part of the image shift range may be consumed by the offsets.
Even when this option is off, the Navigator will use the calibrated offsets wherever appropriate to make features marked at one magnification appear to be at the same stage coordinates at another magnification. Thus, while calibration of the offsets is necessary for optimal performance of the Navigator, the use of this option is more of a convenience than a necessity.
Trim dark borders in Autoalign
When this check box is selected, SerialEM will always analyze each image being correlated in an autoalign and exclude dark regions that appear to be outside the area of the beam. Even when the option is not selected, this analysis is done in low dose mode, when performing tasks at lower magnification, and in the first round of the Realign to Item routine (where it is also useful for excluding grid bars). The Tasks menu command Disable Dark Trim in Align can be used prevent this automatic trimming operation in selected cases.
Set Autoalign Trim Fraction
Use this button to set the fraction to trim off each edge of the images that are being aligned. For example, with a fraction of 0.1, 100 pixels will be trimmed off each side of a 1K x 1K image. In rare cases, the default value of 0.04 will not be adequate and a larger fraction is needed to exclude peripheral parts of the image with strong contrast. Because this is needed so rarely, this value is not stored in the settings file between SerialEM sessions.
Erase periodic peaks in Align/Focus
When this check box is selected, the autoalign and autofocus routines will analyze an autocorrelation of each image to find periodic peaks from repeated structures (except when used for Realign to Item in Low Dose mode). If at least 2 peaks are found in each direction (and at least 5 total), they go out far enough from the origin, and the vectors to these peaks match well enough between the images, the program will replace signal with random noise at all of the positions for this regular pattern in the FFTs. This erasing has been found to be effective for preventing spurious alignments on the wrong square with cross-line gratings and on the wrong hole with gold foil grids having hexagonal patterns; it should work as well with regular gold foil grids, but has been found to be unreliable for carbon foil grids having regular hole arrangements. This erasing is done by default when doing image or stage shift calibrations but requires 2.5 times more peaks to be found before erasing is done; this default can be overridden with the property 'NoDefaultPeakErasing' set to 1, in which case the option set here prevails. The option here determines whether erasing is done when running Realign to Item outside of Low Dose mode, but in Low Dose mode, erasing is determined solely by the Navigator menu entry Erase Periodic Peaks in LD.