Fluorochromes: properties and characteristics

Immunofluorescence microscopy provides a sensitive means by which antigens can be localized within tissues or individual cells. For the most effective use of this technique the researcher can draw upon basic information on factors that affect the brightness of the fluorescence image, and how well that image can be distinguished from background fluorescence or interfering fluorescence signals. A wide variety of fluorochromes are available, with emitting wavelengths that range from the blue-violet end of the visible spectrum to the infrared. Individual fluorochromes are characterized by their extinction coefficients, quantum yields, susceptibility to photobleaching, the wavelengths at which they maximally absorb excitatory and emit fluorescent light, and how far apart those wavelength maxima are separated. Additional choices for fluorescent labeling of antibodies are provided by the availability of fluorescent quantum dots. Informed choices of fluorochromes can obviate many problems, particularly with regard to situations in which two or more antigens are to be localized simultaneously within a specimen.