Abstract: This chapter contains sections titled: Overview Applications of Fluorescence Microscopy Physical Basis of Fluorescence Properties of Fluorescent Dyes Autofluorescence of Endogenous Molecules Fluorescent Dyes and Proteins in Fluorescence Microscopy Arrangement of Filters and the Epi-Illuminator in the Fluorescence Microscope Objectives and Spatial Resolution in Fluorescence Microscopy Causes of High Fluorescence Background The Problem of Bleedthrough with Multiply Stained Specimens Quenching, Blinking, and Photobleaching Examining Fluorescent Molecules in Living Cells