STEM Mode

SerialEM supports STEM imaging on Thermo/FEI microscopes through either a Thermo/FEI STEM unit or a Gatan DigiScan unit, and on JEOL microscopes through a DigiScan unit, or through the native JEOL interface on most newer scopes.  The following features are noteworthy, and the links are to help sections with more explanation and detail:

• Basic aspects of behavior in STEM mode, such as beam blanking and image contrast reversal, are governed by options in the STEM Control panel.
• Detectors can be selected for acquisition, and multiple detectors can be acquired from simultaneously to the extent possible.  The Camera Setup dialog allows control of image acquisition and the selection of detectors, as described in the section Controls in STEM Mode.
• Focus can be changed dynamically while scanning to keep a tilted specimen in focus.  With Thermo/FEI STEM, accurate dynamic focusing requires calibrating the timing of the scan for the particular acquisition conditions, as described in Quick Flyback Time.
• On a Thermo/FEI, both nanoprobe and microprobe mode are recognized and can be calibrated separately for effective use.
• With Thermo/FEI STEM, manual focusing must be done only with the objective lens, not with objective and condenser ('IIntensity & Objective' in the TEM User Interface) or condenser only.
• Tilt series can be acquired in STEM mode; aspects that are different from TEM mode are described in Tilt Series in STEM Mode.
• Images from more than one detector can be saved during a tilt series with the aid of the Tilt Series Extra Output dialog.
• To deal with problems caused by tracking and focusing images not being centered on Record images, the shifts between these image types can be calibrated quickly and then applied during a tilt series (see Assess Interset Shifts).
• Low Dose mode can be used in STEM mode, either to reduce specimen exposure or to provide versatile control over magnifications for tracking and focusing.
• Montaging can be done and the Navigator can be used in STEM mode.

Some features do not work in STEM mode: beam autocentering, specimen cooking, assessing angular ranges, and checking autofocus.

The configuration and calibration of SerialEM for STEM mode is described in Setting Up STEM.

Autofocusing

Autofocusing is done by analyzing the power spectra of a series of Focus images.  It proceeds in two phases.  In the first phase, the program does a simple analysis of the total power over a certain spatial frequency range, after subtracting a background power level.  It changes focus in whatever direction increases power over that at the current defocus, and moves in relatively coarse steps until it reaches a focus where power falls off again.  It then takes images at finer focus steps so that there are at least two images on each side of the image with the best focus.  These five images allow the second phase of analysis, in which it derives a measure of beam size for each of the 4 adjacent images by comparing the shape of its power spectrum with that of the best focused image.  This measure varies linearly with distance from focus, so it is possible to fit a pair of lines to the measured sizes and take focus as the intersection between the two lines.  Because of this interpolation method, it is not necessary to search for and take an image at exact focus.

This method requires that there be enough features in the Focus images to give reliable power spectra for the analysis.  The situation is more challenging than for autofocusing in TEM, where a few high-contrast particles on a featureless background can give an adequate cross-correlation for determining beam-tilt-induced image shift.  It is definitely necessary to pay attention to whether the focus area has enough image detail, and it may be necessary to use larger images (center half instead of center quarter for Thermo/FEI STEM, or with the magnification increased by 2 instead of 4 for DigiScan).    Another limitation of the analysis is that the total power measure becomes insensitive for very blurred images, beyond ~5 microns of defocus.  The first phase of the search may thus fail to find a peak in the power within a reasonable number of steps.  When this occurs during tasks, tilt series, or scripts, the program stops with an error message.  When it occurs after autofocus was started manually, you are given the choice of whether to stop (without modifying defocus) or to continue searching from the last defocus tested.

Acknowledgements

The development of these STEM capabilities was partially supported by contributions from owners of JEOL microscopes at the University of Texas, Dallas and the Naval Research Laboratory in Washington, D.C., and owners of a Thermo/FEI microscope at the Weizmann Institute of Science in Rehovot, Israel.  Assistance and access to STEM-equipped microscopes was provided by Chen Xu at Brandeis University, David Morgan at the University of Indiana, and Tomas Molina at the National Center for Microscopy and Imaging Research.