Fluorescently labeled cells and tissues exhibit a characteristic photobleaching curve in response to the excitation by light. A high percentage of the photobleaching process can be attributed to the generation of free radicals. The use of free radical scavengers has been shown to decrease the rate of photobleaching. Common scavengers include n-propyl gallate, p-phenylenediamine and DABCO (1,4-diazobicyclo-(2,2,2)-octane). Live systems have been reported to reduced photobleaching in the presence of vitamin C or Trolox. If possible, liquid embedding media should be used. Samples mounted in liquid media seem not to shrink in volume over time when sealed properly. Shrinking is at least less than in embedding media which harden by drying (like Mowiol, or ProLong from Molecular Probes). Samples embedded in liquid media like Vectashield or glycerol plus DABCO can be stored in the freezer (-20°C).

Cover slips

Most lab suppliers offer only 0.15 mm (Nr. 1) cover slips, which are not adequate for high resolution microscopy. The objectives designed for the use with cover slips are generally designed for 0.17 mm cover slips.

Cleaning

Under certain conditions, some cover slips may show surface fluorescence generated by contaminating material. Clean your cover slips with analysis grade methanol, ethanol or acetone (any of these will probably work) and air-dry the cover slips prior to use.