Improved depth of field in the scanning electron microscope derived from through-focus image stacks

The depth of field limit in the scanning electron microscope (SEM) can be overcome by recording stacks of through-focus images (as in conventional and confocal optical microscopy) which are postprocessed to generate an all-in-focus image. Images are recorded under constant electron optical conditions by mechanical Z-axis movement of the sample. This gives rise to a change in magnification through the stack due to the perspective projection of the SEM image. Calculation of the necessary scaling as well as the derivation of best focus information at every patch in the image--and a contour map function derived from the selected patch depths--are incorporated in a new software package (Auto-Montage Pro). The utility of these procedures is demonstrated with examples from the study of human osteoporotic bone, where results show uncoupling of resorption and formation. The procedure can be combined with pseudo-colour coding for the direction of apparent illumination when using backscattered electron (BSE) detectors in contrasting positions.