Scanning Transmission Electron Microscopy Facility
STEM Operation and Methods
The STEM is operated at 40 keV with a probe focused to 0.25 nm. The sample is maintained at -150°C to
eliminate contamination and to reduce mass loss.
The scan raster is 512 x 512 points with a dwell time of 30 µs per pixel (8.5 sec per scan). The
area to be scanned is selected from a low magnification (low dose) image and scanned only once.
Focusing is done on an adjacent area prior to the scan. For most mass measurements, the pixels are
separated by 1 nm giving a scan width of 0.512 µm. These scanning conditions usually provide the best
compromise between resolution of the specimen and radiation damage to it.
Electrons elastically scattered through large angles (40-200 mR), through small angles (15-40 mR),
or unscattered (0-12 mR) are collected on separate detectors consisting of scintillators and
photomultipliers. Both large and small angle signals (normalized by the total beam current)
are recorded digitally. The dose is kept below 1000 electrons/nm² to assure that mass
loss from radiation damage is less than 2.5% at the -160°C specimen temperature employed.
Digital images are recorded with the STEM from the annular dark field detectors.
Areas of the grid to be used for mass measurements are selected for clean background
and adequate numbers of both TMV and specimen particles. The background is computed in
clear areas in each image, and the intensity minus background is summed over each particle
and multiplied by a calibration factor to give a mass value.
The STEM calibration factor is checked in each image using TMV which is included as an internal control.
The TMV also serves as a qualitative control for the whole process of specimen preparation. If the
TMV looks good, the specimen has a chance of being good, but if it looks bad, some step in the
specimen preparation has gone wrong. Different kinds of mass measurements can be obtained from
this data. The total mass of the individual particles can be used to determine their oligomeric
state. For filamentous specimens, the mass per length can be used to determine the spacing of
repeating units. Similarly, the mass per area can be a useful measurement on two-dimensional arrays.
Last Modified: June 12, 2009
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