Comparison of 3D microscopy methods by imaging a well characterized test object

We used a 10-μm-diameter fluorescent bead as a test object for 3D microscopy, and independently determined its structure by examining 1-μm-thick physical sections of the bead. Images of the full bead on different 3D microscopes revealed a number of aberrations and distortions. Images also showed evidence for absorption and/or scattering on all confocal and wide-field microscopes tested, but not on a two-photon microscope. The best 3D images of the bead came from deconvolution of either widefield or certain confocal images. For deconvolution of partially confocal data, a region extending 7 μm beyond the top and bottom of the bead contained out-of-focus-light information essential for correct restoration. Fully confocal images required 10fold less computer time for deconvolution, but 1000-fold more excitation light than a wide-field image, even though the resultant restorations were reasonably comparable. Laser imaging of the bead appeared to produce an artifactual image for which we currently have no explanation.