About Low Dose Mode

The essence of low dose mode is that focusing and tracking operations for a tilt series are done in a location separate from the area being recorded, to spare that area from unnecessary beam exposure. In order to provide accurate focusing and tracking, these separate areas must be displaced along the tilt axis from the center of the recording area. SerialEM's low dose mode provides five 'areas' that can be defined to have different settings of magnification, spot size, and beam intensity, centered in up to 3 different locations. They are named for the corresponding camera parameter sets that are used to acquire images from them. The areas are:

Record area - the area where the region being recorded is located. The magnification and beam should be set up for the final image acquisition. Both Record and Preview capture images from this area; the Record camera parameters would be set up for the final acquisition, while Preview parameters would be set up for a highly binned, low exposure image.
View area - this area is centered on the Record area. The magnification should be low (but not in Low Magnification mode) for several reasons: to provide an overview of a larger area, to minimize specimen exposure, and to provide reliable tracking in the various SerialEM 'tasks' that require lower magnification views (but the latter is not essential, as explained below). Taking an image with the View camera parameters will acquire from this area. View camera parameters should be set up for a binned, low exposure image.
Focus area - this area is displaced along the tilt axis from the Record area. The beam must be set up so as not to intrude on the Record area. Taking an image with the Focus camera parameters will acquire from this area.
Trial area - like the Focus area, this area is displaced along the tilt axis and the beam must be constricted. Taking an image with the Trial camera parameters will acquire from this area.
Search area - this area is centered on the Record area and could be set up for even lower magnification than the View area, or with other differences in parameters. Taking an image with the Search camera parameters will acquire from this area. However, the option to Use View for Search in the Camera menu can be used to simplify the situation and acquire with View camera parameters, even though a Search command, button, or script command is used to take the image. The following shows the relationships between Low Dose areas, the buttons or commands used to start an acquisition, and their corresponding camera parameter sets. The bottom part shows the situation when montaging, where options in the Montage Setup dialog control which area a montage is acquired from and the Use View for Search and No Mont. Map Params options in the Camera menu control which camera parameter set is used..

Although the Focus and Trial areas can be set up with completely independent parameters, it is most convenient to constrain them to be identical. This saves the effort of setting up the beam for both areas, avoids the image shift settling time required for getting between them, and minimizes the total range of image shift needed for low dose work. The latter factor is important on the 300 KV Tecnai or Polara because the objective aperture begins to occlude the image area for relatively small image shifts. Image shift is also quite limited on the JEOL 2100/2200 unless the scope has the high power image shift option, although projector shift is usually used for image shift to minimize this problem.

Microscope Parameters Stored. The parameters that can be set separately for each low dose area are the magnification, spot size, beam brightness, filter settings if there is an energy filter. The latter include whether the slit is in, and the slit width and energy loss. On a Thermo/FEI scope, the probe mode (nanoprobe versus microprobe) is also stored. On the JEOL, the alpha setting can also be set separately, although no more than two alpha settings can be used. Absolute beam position is not stored, but a relative beam shift between areas can be set. Diffraction mode can be used for any area, although it is likely to be useful only for the Search area. If diffraction is used, the camera length and diffraction focus are stored as parameters.

Special Features in Low Dose Mode. In low dose mode, numerous features of SerialEM operate differently.

Tasks that use lower magnification tracking (e.g., finding eucentricity and resetting image shifts) will generally use View images, although some have menu entries to control which area is used. However, each such task has a required minimum field of view when run outside of low dose, which sets a minimum magnification at which it should be run. When the View magnification is higher than that minimum, and the Search magnification is lower than View and not in LM mode, the program will use Search instead to minimize the risk of excessive shifts between images. This means that you do not need to set up View to provide an adequate field of view for all tasks, if you have set up Search as a suitable alternative. See the Tasks Use View Even if Search Better command for more details.
Autoalignment operates with two autoalign buffers. A Record or Preview image will be aligned to the image in the first autoalign buffer, whereas a Trial or Focus image will be aligned to the second autoalign buffer. The 'Align to' button in the Image Alignment & Focus control panel will indicate which are the two autoalign buffers.
Autoalignment will work with the beam constricted inside the camera field because autoalign will detect dark areas in the corners of the image (namely, areas outside the beam) and use only the largest rectangle inside these areas for alignment.
The Tilt Series Controller uses D and E as the two autoalign buffers. It uses images of the Trial area for tracking, but it also maintains a reference image from the Record area, so that each new Record image is aligned to that reference and then replaces it.
If you choose to adjust intensities automatically during a tilt series, only the intensity of the Record area will be adjusted.
Since the View and Search areas often have very different magnifications from each other and from the other areas, display zoom will be remembered separately for these two areas. This should make it easier to recognize similar features when toggling between images from the different areas.

Setting Properties of Areas. You can set the properties of a low dose area by making it be the current area then adjusting its features. The current area and its beam properties are displayed on the second line of the Low Dose control panel . There are two ways to set the current area: take an image of the area, or select the area with the 'Go to' button in the control panel. You can adjust most properties of the current area by turning on the 'Continuous update of mag & beam' checkbox. With this option on, any changes in the magnification, spot size, condenser lens setting, or energy filter settings will change the defined properties of the area. However, beam position is still a global feature that applies to all low dose areas, so a change in beam position is not recorded with 'Continuous update' on. (A relative beam position can be set for each area with a separate control.) If the option is off, you can change these features temporarily, but the stored properties of the area will be reimposed next time a picture is taken of the area.

If you want to make initial adjustments to the properties of an area with no exposure of the specimen at all, then select the area with a 'Go to' button and adjust its features.

Setting Positions of Trial/Focus Areas. The positions of the Focus and Trial areas can be set by selecting the Focus or Trial radio buttons in the 'Define position of area' box. Once one of these buttons is selected, two pieces of information become available at the bottom of the Low Dose control panel . One is a text box showing the distance along the tilt axis from the Record area to the area being defined, where negative and positive numbers reflect opposite directions along the axis. The other display shows essentially a safety factor for spillover of the beam from the area being defined into the Record area; it is the distance between a circle circumscribing the area being defined and the edge of the Record area.

There are several ways to adjust the position of the area being defined. In addition to simply typing in a distance in the text box, the most convenient is to take a picture of the View area. The position of the area being defined relative to the center of the View will be shown by a green cross, and you can click with the left mouse button to select a new position. On a View image, the Record area and the area being defined will also be shown with a box, with a circle circumscribing the area being defined. However, if Focus and Trial are being kept at the same position, the circle will circumscribe the larger of their two acquisition areas. They can be kept together either with the option Keep Focus and Trial identical option in the Low Dose control panel or with the specialized option in the Tasks menu, Keep Focus & Trial at Same Position. In the latter case, the magnifications of the two areas are taken into account when picking which area to circumscribe.

Offsets. Several different "offsets" can be set by different procedures:

A defocus offset can be applied when going to the View area in order to increase contrast for the View images. The value can be set with a spinner in the 'Offsets for View' box of the Low Dose control panel.
The View area can be centered on the Record area by recording a shift offset for the View area. This is done by centering a feature in Record, then taking a View image and shifting that feature to the center; see Shift offset for View for details.
Beam shifts can be imposed to center the beam independently in each area using controls in the 'Additional beam shift' box. The procedure is to center the beam in the Record area then go to other areas and record the additional beam shift required to center the beam by checking and unchecking the 'Set' box. See Additional beam shift for details.
Added beam tilts can also be recorded at the same time as the beam shift, if this enabled by a property Setting. On a Thermo/FEI scope, one can also impose a dark field beam tilt for the Trial and Focus areas, which might be a way to keep the beam from being occluded by the objective aperture.

Balancing Shifts. The final concept that needs explaining is that of balancing the shifts. On the 300 KV Tecnai, one should think of the objective aperture as defining a circle within which the specimen can be imaged by means of image shift. If the Record area is in the middle of this circle, then the Focus and Trial areas will be displaced to near the edge of the circle, and there may not be much range of image shift left for tracking during the tilt series. Given these limitations, it is preferable to arrange the Record and Trial areas so that the center of the circle is midway between them, because then neither one will be as close to the edge of the usable circle as the Trial area is with the Record area in the center. This is accomplished by pressing the 'Balance Shifts' button. The change can be undone by pressing the 'Center Unshifted' button, so-called because it will recenter the areas that are considered unshifted (Record and View).

Your low dose parameters are saved to your settings file and will be restored when you restart SerialEM. If you have never started low dose before, the areas are undefined, and they will acquire the current microscope settings when they are first activated. Three sets of parameters are stored: one for a non-energy filter camera, one for an energy filter camera, and one for STEM.

Steps for Initially Setting up Low Dose

It is somewhat difficult to get low dose set up initially, but following a set procedure may be helpful.

Before going to low dose mode, set the magnification and beam strength that you will want for the View area.
Turn on low dose mode and take a picture of the View area.
Turn on 'Continuous update' to adjust the View area properties further. Be sure to set any View-area-specific energy filter settings.
Copy View properties to the Record area with the 'R' button in 'Copy current area mag & beam' radio group.
Take a Preview or Record picture to go to the Record area, then turn on 'Continuous update'.
Adjust the magnification for the Record area, and any specific filter settings.
Adjust the beam strength by taking a Preview or Record and selecting 'Set intensity' in the Tasks menu. Enter the desired number of counts in a Record image. If there is a big change, this may take 2-3 iterations.
If the beam is too small, reduce the spot size; if it is too large (i.e., you are concerned about extra exposure to the Trial/Focus areas), then increase the spot size.
If you want to get the beam to be not much bigger than the camera field of view, you need to condense it so that you can see the whole beam, then expand by a controlled amount. See step 14.
Make sure 'Keep Focus and Trial identical' is selected and copy Record properties to the Trial area with the 'T' copy button.
Select the radio button to define the position of the Trial area.
Take a View picture. Click with the left mouse button to define a position for the Trial area. Note the 'Maximum area separation' and click again to get a sufficiently positive separation, or just type in a position.
Take a Trial picture. Adjust area-specific filter settings (e.g., slit out).
Here it is essential to get the beam constricted to just the Trial area and centered on it. You can do this either by adjusting brightness and position blindly or by using the 'Set Intensity' and 'Move Beam' operations in the Tasks menu. When using 'Set Intensity', note that changing intensity by a certain factor should change beam diameter by the square root of that factor. Also note that your changes will not be accurate when the beam does not fill the camera field. In general, the procedure is:

Constrict the beam enough so that you can see enough of its edge to be able to center it.
Change spot size if necessary to get close to the desired intensity.
Center the beam.
Expand the beam until it just circumscribes the area.

If you are restarting with some existing parameters, you would not do all of these steps. You would probably take a View and adjust its properties if necessary (steps 2 and 3), take a Record and adjust its properties (steps 5-9), then adjust the Trial/Focus area (steps 11-14).