Eucentricity by Focus Setup Dialog
This dialog lets you make calibrations needed for the Eucentricity by Focus routine and set a few parameters controlling how the procedure runs. The routine finds the eucentric point by setting a particular absolute focus value (objective lens strength), measuring defocus with beam-tilted images, and adjusting the Z height to bring the measured defocus to the same same value that it had when the microscope was eucentric with that same absolute focus value. (This procedure has been commonly referred to as 'Z by G' and Chen Xu has distributed a script by that name.) The point of the calibration procedure here is to measure the defocus and read the absolute focus from the microscope when the specimen is known to be eucentric. This measurement can be done at a preferred defocus and/or with a defocus offset to make it more reliable. Before the calibration, the stage is moved in Z to establish backlash from below if necessary. The Eucentricity by Focus routine itself does all Z moves with the same direction of backlash.
With an appropriate calibration, the Eucentricity by Focus routine can find eucentricity from a large distance away in Z, and can also do so with very large defocus offsets. However, in either of these cases, an autofocus calibration over a wide range will be needed. See Extended Autofocus and Non-reciprocity Calibration for Using Eucentricity by Focus for details.
The relation between defocus at eucentricity and absolute focus is magnification dependent, so separate calibrations are stored for different magnifications. At a given magnification, there can also be separate calibrations for different cameras, and for three different states: Low Dose Focus, Low Dose View, and not in Low Dose mode. These calibrations are stored in the settings file, not in the calibrations file, because they may need to be redone frequently (e.g., for each session).
To make the defocus measurements as reproducible as possible, the illumination conditions are stored (intensity, spot size, and probe mode or alpha) as well as the defocus offset and beam tilt used in the measurement. The line of the dialog below the two 'CALIBRATE with' buttons shows the current camera and which of the three Low Dose states applies. Below that is a summary of the relevant conditions. On the left are the current values from the microscope and program. On the right, if there is a stored calibration matching the magnification, camera, and Low Dose state, are the stored values. You can run the calibration either with the current or the stored scope settings. Here are some relevant points:
Controls Relevant to the Calibration:
CALIBRATE with Current Settings
Press this button to run a calibration with the current settings, possibly overridden by entries below for focus offset and beam tilt. The calibration will simply measure defocus 3 times and store the average result. If there is an existing calibration shown, it will be replaced by the new one; otherwise, a new calibration is stored.
CALIBRATE with Stored Settings
This button is enabled if there is a calibration shown for the current conditions, except that it will be disabled when not in Low Dose mode if the spot size or probe mode or alpha do not match the current conditions. Press it to run with the stored settings, possibly overridden by entries for focus offset and beam tilt. The calibrations will replace the existing one.
Calibrate with focus offset of
Turn on the check box and enter an alternative value for the focus offset to use in a calibration. For measuring with View, this offset overrides both the View defocus offset and the autofocus focus offset. This setting is very important when using View: it is recommended that you not use a defocus offset bigger than -50 if possible, to avoid the need for an autofocus calibration extending to very high underfocus values.
Calibrate with beam tilt of
Turn on this check box and enter an alternative value for the beam tilt to apply when measuring defocus. You may well want a higher value for focusing with View, to yield larger displacements that can be measured more accurately.
Controls Relevant when Running Eucentricity by Focus:
Use View area in Low Dose mode
The checkbox controls whether the Eucentricity by Focus routine will measure defocus with View or Focus in Low Dose mode. There must be a calibration for whichever area will be used.
Use current area - 1/4 - 3/8 - 1/2 field
These radio buttons are enabled when using the View area and allow you to measure defocus with a selected subarea, to minimize the contribution from regions several microns from the center that may be at a different height. The closest feasible region for the particular camera
Maximum total change in Z
The Eucentricity by Focus routine will stop with an error and restore the original Z position if the change in Z implied by the latest defocus measurement would result in the total change in Z exceeding this amount. This entry is intended to provide some protection against wild defocus values being measured on a bad specimen area. However,because of the nonlinear relationship between Z and defocus, the entry may need to be considerably larger than the actual Z change for it not to fail on large Z changes. This depends on how well the nonlinearities are accounted for in the procedure (see below). For example, an entry of 140 may be needed to allow for 100 micron changes.
Threshold Z change for iterating
The routine will iterate up to 5 times as long as the defocus difference from the target value is bigger than the threshold value set here. The default value is 0.5 microns; a smaller value might be appropriate if stage moves are quite accurate, whereas a larger value might be needed if they are relatively inaccurate.