Generation of virtual 3D images with every light microscope or scanning electron microscopy (SEM)

Abstract

A major issue of microphotography is the limited depth of focus. This problem can be overcome by taking series of images (z-stacks), which are processed by focus-stacking software to extract a sharp image with an infinite depth of focus. Furthermore, a depth map describing the z-positions of the sharpest pixels is produced, that can be used to generate virtual 3D images. Here we describe the freeware program PICOLAY and its use with light- and scanning electron microscopy (SEM). For SEM, we show different ways to produce stereoscopic images, based on only two or even a single image.