chapter  3
14 Pages

Fluorescence and Fluorescence Microscopy

Fluorescence microscopy is the definitive technique of cell biology. The histologist uses conventional transmitted light microscopy, and the anatomist uses electron microscopy, but the cell biologist uses fluorescence. The ability to

label

individual structures, molecules, or cell compartments gives us enormous power to visualize the structure, and even the dynamics, of the workings of cells. Immunolabeling identifies components with exquisite sensitivity in fixed (and therefore dead) tissue, whereas tagging with GFP

and other expressible fluorescent proteins (Chapter 12) can reveal molecular information about living cells. Fluorescent in-situ hybridization (FISH) reveals genes. Add to this the dyes such as DAPI (DNA) and rhodamine 123 (ER), which target specific cell structures and compartments; indicator dyes that can reveal ion concentrations and membrane potential; and the natural fluorescence of many important cell components (NADP, chlorophyll), and the fundamental power and importance of fluorescence microscopy are clear. Fluorescence microscopy also provides the underpinnings of more recent techniques, such as confocal and multiphoton microscopy, which now enable us to image in three dimensions, not just two.