This dialog box allows you to set the beam intensity and other conditions for centering the beam automatically. Essentially, you need to set the beam to a size appropriate for the method of autocentering to be used, then you may need to adjust the exposure time and binning to avoid saturating the camera. You can take pictures from within this dialog to check both of these adjustments. The autocentering routine can use two methods: finding the edges of the beam, as the Center Beam command does, or simply measuring the average location (centroid) of all the counts in an image containing the whole beam. When the beam edges are sharp enough, which may be the case only for FEG microscopes, the edge-finding method is preferred because the centroid method requires a greater intensity change and requires two images to avoid an error if part of the beam is out of the image. To use the centroid method, you need to make the beam substantially smaller than the camera field, with the diameter perhaps half to two-thirds the size of the camera. The centroid will also be off from the beam center if the sample causes large gradients in intensity.
The edge-finding method can be applied with or without shifting the beam to bring a large portion of edge into view. If no initial shift is applied, the beam needs to be adjusted so that you can see a substantial fraction (1/3 to 1/2) of its edge in the four corners of the field. If the beam is not reliably close to circular, it should be adjusted until it is just inscribed in the camera field of view; there should be no need to make it smaller than that.
With an initial shift applied to bring the beam edge to the center of the field, the beam can be larger than the camera field, perhaps up to twice as big. This option provides one solution if the beam shifts its position when condensed small enough to use it without any shift. See the description of this option below for details.
The program can store the settings to control the autocentering separately for each spot size, magnification, and camera. In addition, separate settings can be stored for intensities on either side of condenser crossover. This does not mean that you always need to set up parameters for every set of conditions. To a limited extent, the program will derive settings for a particular mag and spot size from the settings for a nearby mag and spot. However, it cannot transfer settings from one camera to another or from one side of crossover to another. In Low Dose mode, there is an option to constrict the beam by a specified amount from whatever the current settings of the Trial area are when autocentering; this avoids having to save specific intensity values that may need adjusting, and should be more convenient in many cases.
This dialog is non-modal (as of SerialEM 3.8), which means other operations are allowed while the dialog is open. Three incompatible operations are prevented when the dialog is open: changing the current camera, turning Low Dose mode on or off, and beam autocentering itself. Other tasks and complex operations will temporarily disable all the controls in the dialog.
There are some fundamental differences in the way the dialog behaves and how you can adjust settings depending on whether Low Dose mode is on or off.
With Low Dose mode off, beam adjustment is enabled by turning on the option Setup beam on scope and track scope state, which links the settings shown in the dialog to the state of the microscope. When the option is turned on, the microscope is switched to the state specified by the dialog parameters. Changes in the dialog will be imposed on the microscope; changes on the microscope will be reflected in the dialog. The Bigger and Smaller buttons will be enabled to adjust beam size, and the Test Acquire button is enabled because the scope is already in the needed state. Changing the spot size, magnification, or probe mode in either the dialog or the microscope will switch the dialog to a different parameter set, and scope intensity will be set to the intensity setting of the new parameter set.
You can set up these parameters by adjusting the beam to a likely size on the viewing screen, then taking an image with Test Acquire. Adjust the beam size as needed using the buttons or the Set Intensity command in the Tasks menu, and reduce the exposure time or binning to avoid camera saturation. These latter values are originally based on the Trial camera parameter set.
In Low Dose mode, autocentering images are taken with the Trial area microscope parameters, and only the intensity can be changed from the current Trial microscope state. Here there is a basic choice: whether to save specific intensity and acquisition values for particular magnification and spot size combinations, or whether to use the simplified approach of taking Trial images with a specified reduction in beam size. In the simplified method, no parameters are stored except the percentage reduction in beam size. When autocentering is run, the current Trial exposure time will be used, and the current binning will be reduced by one step if possible. This method does require the beam intensity to be in the calibrated range, unless zero reduction in beam size is specified.
When this simple approach is not used, there are a number of points to be aware of: Here are points to be aware of:
Dialog Controls
Use Trial in LD with beam smaller by x%
Select this option to use the Trial parameters for autocentering, whatever they are at the time, but with intensity changed to reduce the beam diameter by the selected amount. All of the values shown below in the dialog are what would be used if autocentering is done with the current Trial parameters. They are not stored values, and if Trial parameters are changed, the new values would apply instead. Note that the Smaller and Bigger buttons are disabled; only the spinner on this line can be used to adjust the intensity used.
Source of settings
When not using the simplified method in Low Dose mod, this line reports whether the currently shown settings are stored values specific to this spot size and magnification, or whether they have been derived from a different spot size and/or magnification. If settings are derived, then as soon as you modify them in any way (i.e., change intensity, binning, exposure, or centering method), they will be stored and shown as directly set. When using Trial in Low Dose with a smaller beam, the line will either state that the intensity value shown is derived from the current Trial intensity and the size reduction, or will indicate whether the end of the calibrated range of intensity would be reached.
Setup beam on scope and track scope state
As explained above, this option appears only outside of Low Dose mode and sychronizes the dialog and microscope states. With the option off, you can change the mag or spot size selectors to look at different parameter sets without affecting the microscope; you can also change binning or exposure time but not intensity.
Spot size and Mag readouts and selectors
The controls let you see the current magnification and spot size of the dialog, which determine which parameter set is displayed. On Thermo/FEI scopes, the probe mode will be shown as 'uPr' or 'nPr' after the spot size spinner. Outside of Low Dose, you can also change these parameters with the spin buttons. If you want to use these buttons to browse through the parameter sets and possibly delete some, you should open the dialog with Low Dose mode off. Even the sets added in Low Dose mode will be displayed.
Switch to selected mag for autocentering
This option has an effect only outside Low Dose mode. Select it to have the program change to the selected magnification before doing the autocentering. This magnification would generally be the current one when adjusting the settings. If the option is checked, this magnification is recorded when the dialog is closed with the OK button. If the option is already selected when the dialog is opened, the program will switch to that magnification immediately, and restore the magnification when the dialog is closed. Centering at a lower magnification might be necessary for a non-FEG microscope.
Intensity readout
The intensity setting in the current parameter set is displayed but can only be modified when the scope state is being tracked.
Smaller - Bigger
The buttons will decrease or increase the beam diameter by about 10%. For smaller changes, use the Set Intensity command in the Tasks menu or the intensity spin button in the Microscope Control panel.
Binning
Use the spin buttons to set the binning of the exposure. Binning can be reduced to avoid saturation from a bright beam when exposure time is already as low as feasible.
Exposure
The exposure time is shown and can be changed in this text box.
Beam centering method
These radio buttons allow you to choose between finding edges and using centroids, as described above.
Find edges with beam shifted
Use this option to shift the beam before finding the edges, which allows a beam larger than the field of view to be used for centering. This provides a way to avoid problems with the beam being constricted so that enough edges show, such as the beam shifting when condensed that much. Only a single shift is done, not a series of shifts to find a badly mis-centered beam. The shift should be enough to bring the edge of the beam near the center of the field. If the beam is near circular, the center can be estimated reliably from less than half the circumference, but a non-circular beam will definitely give a biased result when the fraction of beam edge analyzed is not close to half. In practice this may be acceptable. This method will also not tolerate the beam being mis-centered by as much as it can be with an unshifted, constricted beam, but it should be able to handle the beam being mis-centered by up to a quarter of the field (compared with at least half the field when using an unshifted, constricted beam. With Low Dose mode off, the beam is shifted on a diagonal to maximize the beam edge in the field; in Low Dose mode, the beam is shifted perpendicular to the axis between Record and Trial to avoid impinging on either the Record area or another more distant area. The shift can be either negative or positive and determines the direction of movement.
Record the shift
This feature can be used to leave the beam miscentered by a desired amount after centering it. This would provide a second way to deal with a consistent shift between the beam used for centering and the uncondensed beam, but it can also be used to bias the position of the beam away from the Record area for an extra margin of safety. It works like the Set button in the Low Dose panel: the program will keep track of the beam shift from when you turn on the checkbox to when you turn it off. After you turn it off, the shift will be shown to the right of the Reset button, in microns on the camera.
To correct for a consistent shift in the condensed beam used for autocentering:
To mis-center the beam deliberately, you need to follow the same steps if you want to see the beam with the screen down or in continuous mode, except that step 4 would be to mis-center the beam. If you want to take single-shot images and move the beam in steps manually or with the Microscope Control panel buttons, you can omit step 3 in Low Dose mode.
Reset/Abort
When not recording a shift, this button is labeled 'Reset' and will clear out a recorded shift. When recording a shift, it is labeled 'Abort' and lets you stop recording a shift without losing the previous value.
Center twice if beam is moved > X um
Select this option to run another iteration of the centering when the first image leads to a beam movement greater the distance specified in the text box. This option is enabled only when using beam edges for centering, because it already runs twice when using the centroid. If the autocentering tends not to come out well enough, either because the beam calibration is not accurate enough at the conditions used, or perhaps because the beam edge was visible only on one side, iterating can give a much more accurate result. For example, if the error in movement is 10%, the centering might only be off by ~1% after iterating. Unlike all the other settings above, this is a global setting that applies to all autocentering parameter sets.
Test Acquire
Use this button to take an image with the current settings and see how large the beam is and whether it is saturating the camera. The screen will be restored to the state it was in before taking the picture unless you are in Low Dose mode.
Delete Settings
The button can be used to delete stored settings, which might be useful if you need to change settings substantially and want to start with one derived from another mag or spot size where you have already changed the settings.