Biology Department Biology Department  

Scanning Transmission Electron Microscopy Facility

STEM Operation and Methods


Probe The STEM is operated at 40 keV with a probe focused to 0.25 nm. The sample is maintained at -150C to eliminate contamination and to reduce mass loss.
Scan The scan raster is 512 x 512 points with a dwell time of 30 s 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 m. These scanning conditions usually provide the best compromise between resolution of the specimen and radiation damage to it.
Detectors 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/nm to assure that mass loss from radiation damage is less than 2.5% at the -160C specimen temperature employed.
Image 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.
Calibration 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.


Top of Page

Last Modified: June 12, 2009
Please forward all questions about this site to: Kathy Folkers

Visit our Anniversary Website One of ten national laboratories overseen and primarily funded by the Office of Science of the U.S. Department of Energy (DOE), Brookhaven National Laboratory conducts research in the physical, biomedical, and environmental sciences, as well as in energy technologies and national security. Brookhaven Lab also builds and operates major scientific facilities available to university, industry and government researchers. Brookhaven is operated and managed for DOE's Office of Science by Brookhaven Science Associates, a limited-liability company founded by the Research Foundation for the State University of New York on behalf of Stony Brook University, the largest academic user of Laboratory facilities, and Battelle, a nonprofit, applied science and technology organization.

Privacy and Security Notice  | Contact Web Services for help