Chapter one - Fluorescence Microscopy

Abstract

This chapter discusses the use of fluorescence in microscopy. As a tool in microscopy, fluorescence provides a number of possibilities in addition to absorption methods. Fluorescence probes, for instance, can be selectively excited and detected in a complex mixture of molecular species. It is also possible to observe a very small number of fluorescent molecules: approximately 50 molecules can be detected in 1 μm3 volume of a cell. Fluorescence microscopy offers excellent temporal resolution because events that occur at a rate slower than about10-8 s can be detected and measured with appropriate instrumentation. When confocal laser scanning is used in fluorescence microscopy, the theoretical limits of the spatial resolution can be obtained in practice. Immunofluorescence microscopy has been the most common application of fluorescence microscopy in cell biology. Fluorescence microscopy is often used for the study of living cells. A large number of cells can be analyzed in a relatively short time by using several fluorescent probes in the fluorescence microscopy technology. The chapter provides details on fluorescence microscopy as well as its uses and advantages.