Classical fluorphores have been widely utilized in biological research, and have been extensively modified to promote particular qualities. Here we review practical methods of optimizing fluorescence signals for microscopy, discuss experimental solutions to common laboratory applications of fluorescent probes, and describe advantages of the sulfonated rhodamines.

Fluorescence output of dyes The usefulness of a fluorescent probe depends critically on how bright it is and on its ability to resist photobleaching.

The brightness of a dye depends on two factors: first, its efficiency in capturing excitation photons, called the molar extinction coefficient, and second, on the number of photons emitted for each photon absorbed, called the quantum yield. The product of these two factors describes the brightness of a dye. Both the molar extinction coefficient and the quantum yield are constants under specific environmental conditions. Consequently, this is a measure that is useful to compare the brightness of dyes.