Digitized Fluorescence Microscopy in Cell Biology Applications

Abstract

Fluorescence microscopy is a widely used technique for studying the location and amount of certain components on or within cells. Specific cellular components can be visualized by linking fluorescent dyes to exogenous macromolecules such as antibodies (immunofluorescence) and ligands, or by producing fluorescent analogs of small molecules normally found within cells. In the past, these approaches have involved fixation of cells and the use of high intensity excitation to image the specimen and produce photographs. More recently, low light level video cameras coupled with frame rate video digitizers have been employed to capture cellular images from the fluorescence microscope. The current and growing appeal of digitized fluorescence microscopy (DFM) in cell biology lies in its ability to quantitatively image the distribution of single classes of macromolecules, molecules and ions in single living cells. In this sense, it is really a new form of optical microscopy for living cells in which the image produced gives directly the distribution of the component probed for. In other forms of live cell light microscopy, we must infer, from other information, the composition of the structure visualized in the image, since we have no a priori knowledge of the molecular structure producing contrast.