Multifluorescence Labeling and Colocalization Analyses

The fluorescence labeling technique is a method with a high degree of specificity and sensitivity and is often chosen as a tool in the study of protein expression and subcellular compartments (1 ). Recently, a large number of fluorescent dyes with distinct fluorescence excitation and emission spectra have been engineered to be used in multilabeling and co-localization experiments. Some of the most common fluorescent dyes are fluorescein isothiocyanate (FITC), Cy2, Cy3, Cy5, TRITC, and rhodamine. These dyes can be excited independently using different laser wavelengths and observed in separate fluorescent channels. The efficiency of the fluorescent probes is, however, hampered by a variable degree of spectral overlap, low quantum efficiencies and extinction coefficients, or rapid photobleaching.