Overview of Fluorochromes

Since its inception in the 1940s, the technique of immunofluorescence microscopy has provided a sensitive, high-resolution method for determining the presence of, and distribution of, an antigen within a specimen. Fluorescent molecules, termed fluorochromes, can be conjugated directly to antibodies by covalent linkage, or coupled indirectly to antibodies via conjugation to proteins A and G or through an avidin-biotin bridge (see Chapters 6, 7, and 25). A fluorochrome coupled to an antibody or other probe may be termed a fluorophore; here, the term fluorochrome is used throughout. The basic features of immunofluorescence are straightforward, but a working knowledge of the commonly used fluorochromes is of value in obtaining maximum performance from immunofluorescence microscopy and flow cytometry. The discussion below focuses on fluorochromes commonly used for immunolabeling, and does not encompass fluorochromes that provide molecule- or organelle-specific labeling without the use of antibodies.