The use of thick specimens (0.5 μm to 5.0 μm or more) is one of the most resourceful applications of high-voltage electron microscopy in biological research. However, the energy loss experienced by the electron beam in the specimen results in chromatic aberration and thus in a deterioration of the effective resolving power. This sets a limit to the maximum usable specimen thickness when investigating structures requiring a certain resolution level.An experimental approach is here described in which the deterioration of the resolving power as a function of specimen thickness is determined. In a manner similar to the Rayleigh criterion in which two image points are considered resolved at the resolution limit when their profiles overlap such that the minimum of one coincides with the maximum of the other, the resolution attainable in thick sections can be measured by the distance from minimum to maximum (or, equivalently, from 10% to 90% maximum) of the broadened profile of a well-defined step-like object placed on the specimen.
