Fluorochromes, unlike classical tissue stains, re-radiate the absorbed light, however, with longer wavelength (red-shifted). Each fluorochrome has a characteristic excitation spectrum which represents the correlation between the wavelength of the excitation and its efficiency. Similarly, a fluorochrome is characterised by its emission spectrum which shows the variation in emission intensity over a certain wavelength range. The red-shift of the fluorescence is illustrated by the emission spectrum having its maximum at a longer wavelength than the excitation spectrum. The peak-to-peak difference is referred to as the Stokes shift.

The time it takes for a fluorochrome to emit the absorbed light is on the order of nanoseconds (10-9 s). However, certain substances display a different behaviour where the lifetime of the excited state ranges between milliseconds and minutes. This is called phosphorescence. Both phenomena are categorized as photoluminescence. The stability of the excited state depends not only on the physical properties of the dye but also on its chemical environment. The variance in lifetime can give important information about a cell, which is exploited in a new technology termed FLIM (fluorescence lifetime imaging).
Hundreds of fluorochromes with known excitation and emission characteristics and well-understood biological target structures have been developed. They are often highly specific in targeting their attachment sites which makes them extremely valuable in biological applications. Their function is 1) to identify and localize biological targets (molecules, ions, organelles or other entities) specifically within complex specimens and enable their detection and possibly quantitative analysis or 2) to produce a spectroscopically observable response to a certain stimulus. Fluorophores (fluorescent probes) are either autofluorescent proteins like the green fluorescent protein (GFP) or fluorochromes that are chemically linked (conjugated) to an active substance such as a protein, antibody or nucleic acid in order to selectively stain a targeted substance or subcellular structure.
There are specimens that autofluoresce when irradiated with shorter wavelength light: this is called primary fluorescence. Only the application of extrinsic fluorochromes and the resulting secondary fluorescence of biological samples has transformed fluorescence from a phenomenon of incidental usefulness to the advanced, powerful detection technique applied nowadays to biomedical microscopy.