Chapter 13 Basic Fluorescence Microscopy

Abstract

Publisher Summary This chapter describes the different aspects of basic fluorescence microscopy. There are five major attributes of fluorescence as a tool in microscopy. The attributes include specificity, sensitivity, spectroscopy, and temporal resolution. Immunofluorescence has been the most common application of fluorescence microscopy in cell biology. It combines the specificity, sensitivity, and spatial resolution of fluorescence microscopy with the selective binding of antibodies to restricted regions of antigen molecules termed epitopes. Multiple epitopes can be localized in the same cell, on the same or on different molecules, by choosing fluorophores with different fluorescence colors. Fluorescence microscopy is also an important biophysical tool for studies in living cells and in reconstituted preparations in vitro . Fluorophores or fluorochromes are fluorescent dyes or probes that are added to cells. Fluorophores are chosen or synthesized for particular applications based on several criteria. These criteria include absorption and emission spectra, extinction coefficient, quantum yield, environmental effects, and chemical reactivity.