Visualization of the Microtubules of Glutaraldehyde-Fixed Cells by Reflection-Enhanced Backscatter Confocal Microscopy

Performing reflection-mode (backscatter-mode) confocal microscopy on cells growing on reflective substrates gives images that have improved contrast and are more easily interpreted than standard reflection-mode confocal micrographs (Keith et al., 1998). However, a number of factors degrade the quality of images taken with the highest-resolution microscope objectives in this technique. We here describe modifications to reflection-enhanced backscatter confocal microscopy that (partially) overcome these factors. With these modifications of the technique, it is possible to visualize structures the size-and refractility-of individual microtubules in intact cells. Additionally, we demonstrate that this technique, in common with fluorescence techniques such as standing wave widefield fluorescence microscopy and 4-Pi confocal microscopy, offers improved resolution in the Z-direction.