Low Dose Control Panel

Low Dose Mode

This check box turns low dose mode on or off. When low dose is turned on, the current area is undefined unless the screen is down. When low dose is turned off, the program returns to the Record area.

Electron dose of current area

If electron dose is calibrated, the dose will appear for the current area in the upper right corner of the Low Dose panel. This dose will be updated continuously when the 'Continuous update' option is selected and will also we changed when the exposure time changes for the current area.

Mag & beam settings of current area

The line will show the name of the current area and its magnification, spot size, and brightness setting as given by a condenser lens strength or illuminated area. The line is shown in black when the 'Continuous update' option is selected, to signify that the values can be readily changed. Otherwise the line will be grayed out and be hard to read.

Continuous update (see tooltip)

When this option is selected, any change of magnification, spot size, brightness, diffraction focus in diffraction mode, energy filter settings, probe mode on a Thermo/FEI scope, or alpha on a JEOL scope will immediately change the parameters of the current area. Otherwise, such changes will be lost when a new image is acquired from the given area, except for intensity changes made with Set Intensity or Set Dose Rate in the Tasks menu.  If there is an IDES dose modulator connected, the current percent duty will also be stored as a parameter for the area.  That value can be changed either with the controls in the IDES Attenutation Control Software or with the 'SetEDMDutyPercent' script command.  Beam shift, beam tilt, focus settings in imaging mode, and image shift displacements for a particular area are NOT recorded when this option is on, but can be introduced by other options in this panel.

Usually, if you have already changed some microscope settings, those changes will be incorporated into the current area when turning this option on.  However, when the current area is View or Search and either the probe mode has been changed or the magnification has crossed the LM-nonLM boundary, the program needs to reassert the pre-existing area parameters to prevent defocus from getting off.  For these two areas, these kinds of changes can be made only after this option is turned on.

Define position of area

Select Focus or Trial to adjust the position of these areas relative to the Record area. (If 'Keep Focus and Trial identical' is selected, it does not matter which area is selected here.) With one of these options selected, SerialEM will try to display a marker point on the image in buffer A showing the center of the area being defined. On a View image, it will also draw boxes representing the Record area and the area being defined, as well as a circle circumscribing the area being defined. You can adjust the position of the area in 4 ways:

1. By clicking with the left mouse button in a View image or in an image from the area being defined.  A View image can be in any buffer or even read in from a file; an image of the area being defined must be in buffer A;
2. By drawing a line anywhere in a View image, including one read from a file, by holding down the Shift key and the left mouse button while moving the mouse.  The line defines a vector that specifies a position relative to the image center. In effect, the line is shifted so that its starting point is at the center and its end point is then turned into a marker point that is treated just like in 1).
3. By entering a positive or negative distance in the 'Position on tilt axis' text box;
4. By shifting an image of the area being defined with the right mouse button (although shifting a Record or View will still change image shift as usual);
5. And even by adjusting image shift through microscope controls when the screen is down.

When you click in a View image, the behavior depends on whether the axis between areas is free to rotate away from the tilt axis, with the 'Rotate interarea axis' option. If that option is off, the marked position will be projected onto the tilt axis and that projection will define the distance of the area along the axis.  If the option is on, the marked position will be used as is, changing both the angle of the axis and the location on the axis.  When you click in an image of the area being defined, the axis rotation is not changed, and the clicked point is projected onto the interarea axis.

You should select 'None' after adjusting the area to avoid inadvertent changes, and to restore the expected behavior when setting the marker or shifting an image with the right mouse button. Note that shifting the area being defined would change its offset until defining is turned off.

Position on Tilt Axis

When the position of the Focus or Trial area is being defined, this text box will show the center-to-center separation between that area and the Record area. You can enter a number to set this distance as desired.

Area separation

When the position of an area is being defined, this line shows the minimum separation between that area and the Record area. This separation is the distance between the edge of a circle drawn just around the outside of the area being defined, as defined by its camera parameters, and the edge of the Record area as defined by its camera parameters. If 'Keep Focus and Trial identical' is selected, the larger of the two areas will be used for this computation.

Go to area buttons

These buttons allow you to go to a particular area without taking an image.  The button for the current area is outlined in green.

Additional beam shift (and possibly tilt)

The controls in this box allow you to impose an independent beam shift for each area relative to Record. You can use this to correct for imperfections in beam centering between the different areas, such as due to mag changes or inadequacy in the coupling between beam shift and image shift. You can also use it to shift the beam in the Trial and Focus areas farther away from the Record area than it would be if it were centered. To set beam shift for an area, first be sure that the beam is centered in the Record area, then go to the area of interest, turn on the 'Set' checkbox, move the beam to the desired position and turn off 'Set'. At this point, if beam shift is calibrated, the value of the shift will appear next to the 'Reset' button. This shift is in microns and in specimen coordinates, where X is along the tilt axis. Press the 'Reset' button to return the shift of the current area to zero. 

If the title line of this group box is Additional beam shift (and tilt), a change in beam tilt is also recorded at the same time as a change in beam shift, and it is applied when changing between areas.  (This feature is enabled by setting the property 'LowDoseBeamTiltShifts' in the properties file; as of SerialEM 3.4, it is no longer restricted to Trial and Focus areas). On Thermo/FEI scopes, the beam tilt recorded in this case would be adjusted by the Rotation Center alignment.  The 'Reset' button will return these beam tilts to 0.

For Thermo/FEI scopes, Dark Field beam tilt provides a better way to adjust beam tilt for the Trial and Focus areas; it is available as of SerialEM  3.6 and the group box title will be Additional beam shift (and DF tilt) unless the 'LowDoseBeamTiltShifts' property is set.  In this case, an absolute setting is stored, not a difference between starting and ending values, so the only the dark field state when 'Set' is turned off matters.  This value is not reset when the 'Reset' button is pressed (should it be?), so to clear it out, just turn Dark Field off and turn 'Set' on then off.  This feature may be useful for allowing larger image shift with a small objective aperture on 300 KV Tecnais and Polaras.

Beam shifts and tilts could be set for the Record area prior to SerialEM 3.6 and can no longer be, to avoid inadvertently setting undesired values for Record and reduce confusion.  Existing values from a previous version will be used until they are cleared out with 'Reset'.

Defocus offsets for View and Search

You can use these controls to set an additional underfocus for the View or Search area. Focus will be changed by this amount when going to the View or Search area (e.g., by taking an image) and then will be restored when going to another area. For example, if you have a focus target of -4 microns, set the offset to -8 to get View images taken at -12 microns defocus.  Be sure to align the View or Search images with the offset feature described next if you use very large defocus values.

The defocus offset is applied after going to the magnification of the area; thus it will be incremental to a focus change imposed by the microscope during the magnification change.  In particular, when going between nonLM and LM, the microscope may restore the last defocus seen in the new magnification range, and the offset is then applied to that.

Defocus offsets are disabled in diffraction mode.  An absolute diffraction focus value is recorded as part of the area settings, and can be modified when continuous update is turned on.  This absolute value is restored whenever going back to an area in diffraction mode.

Shift offsets for View and Search

If image features shift when going between the View or Search and Record areas, one of the Set buttons allows you to correct for this shift. There are two methods for setting the shift offset: 1) using image shift to align an image in buffer A with a reference image, which can be done manually by applying a shift with the mouse button or automatically with the Auto button; 2) placing the green marker point at corresponding locations in the images in buffers A and B.   Method 1 is more direct and potentially more accurate, but may be more difficult to do manually.  Method 2 is simple but depends on the accuracy of image shift calibrations and may also depend on the accuracy of rotation angle and pixel size calibrations.  The Set button for View or Search is enabled whenever there is a shifted View or Search in the A buffer, respectively, or whenever there are marker points on images of appropriate types in buffers A and B. However, the Set button should be pressed only if the shift in A brings the image into alignment with a Record or Preview image, or if the markers are indeed on corresponding points.

1) Setting shift offset automatically: When you press the Auto button for View and the difference in magnifications is not too large (20-fold by default), the program will take a Preview then a View image, attempt to align them and set the shift offset, then take another View to refine the offset.  With the Search Auto button, it will take a View if it has a higher magnification than Search, otherwise it will take Preview, then take Search images to align.   Specifically, it will scale down the higher-magnification image and crop out as much of the lower-magnification image as is needed to find the maximum shift with full overlap between the two images.  The cropped image is left in buffer A and the scaled down image in buffer B, so you can toggle between them to assess whether they are correctly aligned.  Image shift is reset at the start if it exceeds 0.5 micron (this threshold can be set with the property LDShiftOffsetResetISThresh).  The alignment is iterated if the image shift of the first alignment exceeds 0.1 micron (a limit set with the property LDShiftOffsetIterThresh). 

When measuring an offset relative to Record, if the difference in magnifications exceeds a limit (a 20-fold ratio by default, set with the property LDShiftOffsetMin2StepRatio), the program will try to do the alignment in two stages. It picks an intermediate magnification that splits the ratio approximately in half, modifying the intensity of one of the Low Dose areas to work at that magnification.  (In case you need to know the details: If the probe mode or alpha values match between Record and the lower magnification area, then it will modify the Record area parameters by spreading the beam.  If those values do not match, it prefers to modify the lower magnification area, but if that area has too high a defocus offset (20 microns by default, set with the property LDShiftOffsetMax2StepFocus), it will modify the Record area instead, and pick as high a magnification as possible (around 15-fold higher than the lower one).  The motivation there is that nanoprobe mode, or an alpha value used at high magnification, may not work well at too low a magnification.)  When the two-step procedure is done, images are left in 5 buffers: the extract from the View or Search image in A, the scaled down image from the intermediate magnification that extract was aligned to in B, the full View or Search image in C, the extract from the middle magnification image in D, and the scaled down Preview image in E.  Thus, you should be able to assess the alignment at both stages by comparing A with B and D with E.  The biggest problem with this procedure is that the beam edge may appear at the intermediate magnification if the beam is constricted too much or not centered well enough.  This can easily make the correlation align the beam edge to dark material at the lower magnification.

The Zero button is labeled Undo after setting a shift automatically, and you can use that button to revert to the previous offset if necessary.  Only the last shift done can be reverted.  There are three important points about the automatic aligment:

• There must be a well-defined feature in the higher-magnification image that is unique and easily recognizable in the lower-magnification one, and within the central half of that image (unless the maximum allowed shift has been set differently from the default; see Setup Auto Shift below).  Use the Camera menu item Preview Prescan (accelerator key Alt-C - E, script command 'PrescanWithPreview') to capture a 3x3 montage quickly for finding a good area at high magnification.
• For the two-stage procedure, there must be an intensity calibration to allow one of the Low Dose area parameters to be modified.  A warning will be issued and it will drop back to the single-stage procedure, which will likely not work across very large ranges (e.g., 80-fold), or else you will not be able to tell if it worked.
• Be sure to find the View offset before the Search offset (but this does not matter if Search is at a higher magnification than View).

2) Setting shift offset with an alignment shift: The procedure for setting a shift for View is:

1. In the Image Alignment & Focus panel, turn off 'Move stage for big mouse shifts' if it is on.  (This step is optional if you are making a small adjustment to an existing alignment, or if you shift the image in multiple steps each too small to trigger a stage move.)
2. Take a Record or Preview image and use the right mouse button to center a feature that will be recognizable in the View image. Take another image to verify that it is centered.
3. Take a View image. Use the right mouse button to center the feature.  Do not take another image.
4. Press Set. The shift will be recorded as a correction, and the program will take a new View image, unless you skipped step 1 and your mouse shift triggered a stage move.  (In that case, the offset is correctly recorded, but you will receive a message that you need to reset image shift and start with a new Record image in order to check the offset.)
5. If the new View image is not centered (such as if you are using a large defocus offset or the image shift calibration is not good), center the feature and press Set again. The new shift will be added to the existing one.  Repeat if necessary.
6. Turn 'Move stage for big mouse shifts' back on if desired.

The procedure is the same for setting the offset for Search, although here you may want to use a View image instead of Record or Preview as your reference.  If so, be sure to align View with Record first.

3) Setting shift offset with corresponding marker points: The images from a reference area and from the area whose offset is being set can be taken in either order.  To minimize the effect of inaccuracies in the calibrations, try to pick the points near the center of the images, especially for the reference image at higher magnification.

• To set an offset for View, you can have a View in A and a Record or Preview in B; or you can have the View in B and the Record or Preview in A.
• To set an offset for Search, you can have a Search in A and a Record, Preview, or View in B; or you can have the Search in B and the reference image in A.
• If you use a View as a reference image for setting the Search, set the View offset first.
• Alignment shifts in the image buffer are not allowed with this procedure.  The Set button will be enabled even if there are such shifts, but pressing it will bring up a message telling you to clear the shifts with the Clear Alignment button in the Image Alignment & Focus control panel.  If the situation is ambiguous and the image in A could be used to set the offset by method 1, then the message will give you that choice.

General points: Once an image has been used for setting the offset, it cannot be used again; you need to acquire new images.

If for some reason this shift offset becomes inappropriate, you can set it to zero with the Zero button.  However, do not zero out the shifts after taking images for setting the shift and before setting the shift with them.

If the image shift offsets between magnifications are calibrated, this adjustment will work together with that calibration. You can set this adjustment with the option to 'Adjust image shift between mags' either on or off. Once you have set an adjustment, it should work equally well if you turn 'Adjust image shift between mags' on or off.

Setup Auto Shift