MEASUREMENT OF HYDRATED AND DEHYDRATED CRYOSECTIONS BY EELS

Electron energy-loss spectroscopy (EELS) provides a useful method for determining the thickness of frozen-hydrated and dehydrated cryosectio ns in terms of the inelastic mean free path. Cryosection thickness is an important parameter because plural inelastic scattering limits the sensitivity of elemental microanalysis based on core-loss EELS, and be cause overlapping structures can affect interpretation of microanalyti cal data as well as the quality of electron images. The purpose of thi s work was to establish the minimum practical thickness for cutting cr yosections and to explain the measured values for hydrated and dehydra ted specimens. Hydrated sections were typically found to be between 1. 5-2.5 times thicker than expected from the nominal microtome setting; this difference can be largely explained by compression during cutting . Comparison of micrographs from hydrated and dehydrated cryosections of rapidly-frozen, vitrified liver revealed a lateral shrinkage of sim ilar to 20% on drying. The measured compression and shrinkage factors are consistent with dark-field scanning transmission electron microsco py (STEM) mass measurements on freeze-dried sections. Freeze-dried cry osections, cut to a nominal thickness of 90 nm and supported on thin F ormvar/carbon films, had a relative thickness t/lambda(i) in the range of 0.5 for cytoplasm to 0.9 for mitochondria when analyzed at 100 keV beam energy. Mass loss of similar to 30% occurring at high electron d ose enabled useful core-loss spectra to be recorded even from high-mas s compartments such as mitochondria without excessive plural scatterin g. (C) 1996 Wiley-Liss, Inc.