Immunofluorescence: quantitative considerations

The combination of the specificity of the antigen-antibody interaction with the sensitivity of fluorescence detection and quantitation yields one of the most widely applicable analytical tools in cell biology. Immunofluorescence (IF) signals can be measured with microscopes or flow cytometers. Choice for either of the two systems depends on the type of preparation (cells, tissues, etc.), on the type of required information (morphology, distribution, quantitation) and on the number and quality of the samples. In IF a choice has to be made for the most appropriate fluorochromes, reagents, equipment and preparative procedure, respectively, the right balance between lightsource, objective, eye pieces and filters should be sought in microscopy as well as in flow cytometry. The quantitative influence is reported of each of these variables on the eventual fluorescence intensity. New developments are appearing in IF technology. These include laser-scan microscopy, point-addressable optical sensors, phosphorescence microscopy and more sophisticated flow cytometers that allow some morphometry. The possible uses of these systems is discussed.