Chapter VIII-2 - Complementary techniques: Fluorescence microscopy

Abstract

This chapter describes the basic principles of fluorescence microscopy (FM) and provides protocols for its use in the study of invertebrate mycopathogens. Fluorescence is the luminescence of a substance excited by radiation. In fluorescence microscopy, the three possible ways of illuminating the specimen include dia-illumination by a substage bright-field condenser, oblique illumination by a substage dark-field condenser, and epi-illumination by a dichroic beam splitter placed above the objective. The function of the light source is to provide light at a wavelength corresponding to an excitation maximum of the fluorochrome. The role of filters is extremely important in FM. An incorrect selection can cause the background to be too bright to distinguish specific from nonspecific fluorescence and can prevent the use of double-staining. The fluorochromes Calcofluor White M2R, Uvitex BOPT, and Tinopal LPW are widely used to stain fungal cell walls. These dyes bind to sugars in the cell wall and can be invaluable in certain mycological studies. Fungal cells can either be suspended in benzapyrene-caffeine or a drop of this stain can be added to the specimen just before the examination.