Thickness 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 cryosections 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 because overlapping structures can affect interpretation of microanalytical data as well as the quality of electron images. The purpose of this work was to establish the minimum practical thickness for cutting cryosections and to explain the measured values for hydrated and dehydrated 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 approximately 20% on drying. The measured compression and shrinkage factors are consistent with dark-field scanning transmission electron microscopy (STEM) mass measurements on freeze-dried sections. Freeze-dried cryosections, cut to a nominal thickness of 90 nm and supported on thin Formvar/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 approximately 30% occurring at high electron dose enabled useful core-loss spectra to be recorded even from high-mass compartments such as mitochondria without excessive plural scattering.
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Biomedical Engineering and Instrumentation Program, NCRR, National Institutes of Health, Bethesda, Maryland 20892, USA.
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Andrews
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Leapman
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RD
D000818:Animals; D016616:Cryoultramicrotomy; D017348:Microscopy, Electron, Scanning Transmission; D051381:Rats