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J. Biophys. and Biochem. Cytol., Vol 8, 1-11, Copyright © 1960 by Rockefeller University Press

ARTICLE

THE MEASUREMENT OF MASS, THICKNESS, AND DENSITY IN THE ELECTRON MICROSCOPE



R. E. Burge Ph.D.1 and N. R. Silvester Ph.D.1

1 From Wheatstone Physics Laboratory, University of London King's College, London, England

A description is given of quantitative methods using the electron microscope which can be applied to specimens with much smaller dimensions than those which can be used with the established cytochemical methods based on the use of the interference microscope and the techniques of ultraviolet and x-ray absorption. A discussion of electron scattering shows that under chosen operating conditions in the electron microscope the effective total mass-scattering coefficient S of a specimen is almost independent of its chemical composition. An order-of-magnitude agreement is observed at four accelerating voltages between experimental total scattering cross-sections for polystyrene and theoretical values for carbon. The contrast in a micrograph taken under standardised conditions is interpreted in terms of differences in specimen mass-thickness. The measurement of mass, thickness, and density of discrete particles and thin sections in the absence of sublimation is discussed in terms of relevant object models on the assumption of a constant, experimentally determined, value of S. The validity of the proposed methods was examined by measuring the masses of the heads of ram spermatozoa (about 7 x 10-12 gm.) and T2 bacteriophage (about 3 x 10-16 gm.) in the electron microscope. The values agreed reasonably well with those found by interference microscopy and sedimentation-diffusion measurements, respectively. Errors in S and magnification due to contamination and their effects on the results are considered in detail. An application of the methods to a typical electron microscope specimen was demonstrated by measuring the mass of heads of the T2 bacteriophage after staining with uranyl acetate. Errors of measurement are discussed and a minimal measurable mass estimated. Further applications of quantitative electron microscopy are proposed.

Submitted on September 7, 1959


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