Resolution as a function of accelerating voltage in electron microscopy of semithick biological specimens

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Abstract
In the past, biological sections ranging in thickness from 0.10- to 0.50-μm have usually been examined with high-voltage (>500 kV) electron microscopes (HVEM). Now investigators are increasingly using intermediate voltage (200–500 kV) electron microscopes (IVEM), which are more readily available and demand less maintenance. In a study of “typical” plastic-embedded, stained sections of mouse liver ranging from 0.10 to 1.0 μm thick, we determined the resolution obtainable at 100, 200, and 1000 kV. At all three accelerating voltages the resolution (2.7 nm) for 0.10-μm sections was limited only by the sections stain granularity. For 0.25-μm thickness the resolutions were 5.8, 3.1, and 3.1 nm at 100, 200, and 1000 kV, respectively. The maximum usable thickness at 200 kV with resolution sufficient to resolve membranes clearly was between 0.75 and 1.0 μm, depending on the magnification. Resolution at 100 kV was adequate for screening sections up to 1.0-μm thick for preparation defects prior to examination with an IVEM or HVEM.