Mechanisms of Fluorescent Staining

There are several different mechanisms by which fluorescent staining can occur. Some dyes have an affinity for certain substances and accumulate in high concentrations in particular locations or organelles within cells (e.g., lipohilic dyes such as merocyanine 540 accumulate in membranes and reactive dyes such as the succiidimyl ester of carboxyfluorescein diacetate [CFDA] bind cova-lently, predominantly to proteins). Fluorochromes are frequently conjugated to protein ligands (e.g., antibodies), and these conjugates bind specifically and/or in higher amounts to cells expressing the appropriate receptor (or antigen) than to others. Other fluorescent probes may show a marked increase in their quantum efficiency when bound to a particular substance or when they are in a particular environment, a mechanism that was originally termed "fluoro-chroming" or "hyperchroming." For instance, there are not only localized increases in concentration when ethidium bromide (EB), PI, or the Hoechst dyes bind to DNA, but as a result of the change in their environment their fluorescence can increase by up to 100-fold. Similarly, that of the cyanine dyes (e.g., thiazole orange and TO-PRO-1®) can increase by up to 1000-fold when bound to DNA.

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