Comparing methods for 3D microscopy

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Abstract
In previous studies, we examined 3D 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 fluorescence 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 bed containing a much thinner layer of surface fluorescence. In contrast to earlier studies, confocal image of this bead appear to show an accurate thickness for the fluorescence shell. These particular confocal microscopes 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.

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