The Leica confocal microscopy has proven useful for my own work on sub-cellular localization of proteins, mapping projection patterns of individual neurons, development in living embryos, enumeration of flourescent objects, and finally for preparation of high quality illustrations. The improved separation of the signal is obtained at the expense of slightly worse spatial alignment of the two dyes. The files can be merged later with Leica software. UV illumination tends to bleach green dyes rapidly, so only use UV dyes if necessary. I collect the UV scans after the other scans to reduce bleaching.
Different agents works better with different preparations.
Seal the cover slip to decrease exposure to oxygen. Treated cover slips can be stored in the refrigerator for later use For non-adherent cells and small pieces of tissue Place spacers on the left and right sides of the slide or coverslip. Place slide on stage, be sure it is flat. Adjust the pinhole if necessary to increase intensity. Pinhole starts at 1.0 airy unit, which gives good resolution, larger pinholes give brighter images with worse resolution.