Determination of the mass thickness of biological sections from electron micrographs

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Abstract
SUMMARY: Theoretical results are given for the evaluation of the mass thickness of unstained and stained biological sections based on the scattering contrast model for in‐focus electron microscope images. Densitometer measurements on photographic images enable the scattered electron intensity (I) and the incident electron intensity (I0) to be determined, and it is shown that the ratio I/I0 can be related to the mass thickness ρt of a particular region of the specimen by I/I0 = exp (−Spρt). The parameter Sp is given for a range of incident electron energies (E0 = 20–100 keV) and objective aperture sizes (semi‐angle α subtended at the specimen 5–15 mrad). Experimental results on thin stained sections of Bacillus subtilis for E0 = 40–100 keV and α = 3.8–17.5 mrad demonstrate the internal consistency of the theoretical curves for the calculation of mass thickness of both the embedding medium and the biological material. The sensitivity of this method for the determination of mass thickness depends on E0 and α; variations in mass thickness of ±2 mg m−2 can be determined for E0 = 40 keV and α = 5 mrad, becoming less sensitive as E0 and α are increased, so that for E = 80 keV and α = 10 mrad the sensitivity is reduced to ±5 mg m−2. This means that density variations of ±40 and 100 kg m−3, respectively, can be determined in sectioned biological material. Because of the requirement of a linear relation between log (I/I0) and ρt, the theoretical calculations are limited to a maximum mass thickness of 200 mg m−2 or a section thickness of about 200 nm for material of density 1000 kg m−3.These calculations are relevant to applications in microbiology, stereology and autoradiography.