The scanning transmission electron microscope (STEM) can provide low dose, high resolution images of unstained biological samples. Isolated objects or discrete image features can be integrated (after subtraction of substrate) to determine molecular weights and so provide a direct link between biochemistry and electron microscopy. The accuracy of STEM melecular weights improves from about 20% at 10 kD to about 4% at 100 kD and better than 1% above one million D for single particles imaged at a dose of 10 el/Angstroms squared. Practical results on tRNA, nucleosomes, fibrinogen, RNA polymerase, glutamine synthetase, 30s and 50s ribosomal subunits, filamentous viruses and TMV are in good agreement with theoretical predictions. Standard error of the mean can be reduced by averaging over 100-1,000 particles. Histograms of particle mass values can be used to characterize the homogeneity of the preparation. This technique has been applied routinely to a wide variety of users'/collaborators' samples over the past years at the Brookhaven STEM Biotechnology Resource. We propose to continue these productive collaborations and service activities while refining practical aspects of the technique. Further reduction of specimen temperature to 20 K is expected to reduce mass loss below 0.25%/el Angstroms squared now found at 120 K (8x less than at 300 K), permitting higher dose and higher accuracy. Reduction of surface migration at lower temperature is expected to lead to less redistribution of mass during radiation damage. This prediction will be tested using control specimens of known structure (e.g. TMV). A major new thrust will involve use of image averaging programs to improve signal to noise ratio in low dose images. This should permit extension of domainal mass mapping already demonstrated on fibrinogen and dynein to allow detection of the binding of small molecules (1-5 kD) to complexes such as glutamine synthetase and acetylcholine receptor. For particles with spherical or cylindrical symmetry, these programs will also permit determination of mass as a function of radius.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR001777-10
Application #
3103996
Study Section
Special Emphasis Panel (SSS (G))
Project Start
1984-01-01
Project End
1994-03-31
Budget Start
1993-01-01
Budget End
1994-03-31
Support Year
10
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Associated University-Brookhaven National Lab
Department
Type
DUNS #
City
Upton
State
NY
Country
United States
Zip Code
11973
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