Comparing methods for 3D microscopy

In previous studies, we examined three-dimensional images of a latex bead containing a surface layer of fluorescence whose thickness was determined by physical sectioning. Confocal images of the bead from six different microscopes all exhibited a significantly thicker fluorescent shell than actually present. In contrast, deconvolved wide-field images of the bead produced an image with the correct shell thickness. We have now repeated some of these studies using a new latex bead containing a much thinner layer of surface fluorescence. In contrast to earlier studies, confocal images of this bead appear to show an accurate thickness for the fluorescent shell. These particular confocal microscopes (a Biorad 1024 and a Zeiss 510) also yielded essentially aberration free images, which was not the case for some of the earlier microscopes tested. In these recent studies however, deconvolution of the bead appeared less robust than earlier. Of three independent wide-field images of the bead, only one yielded a substantially artifact-free image upon deconvolution.