What are the properties of probes that determine their suitability for FRET applications?

Probes should exhibit strong fluorescence emission upon excitation, allowing for reliable detection and measurement. This includes high quantum yield, which determines the efficiency of light emission upon absorption. Also, the absorption spectrum of the donor fluorophore should overlap with the emission spectrum of the acceptor fluorophore. Three main types of fluorophores have been used as FRET pairs in FRET biosensors: small organic dyes, quantum dots (QDs) and fluorescent proteins (FPs). FPs exhibit high cellular specificity by using tissue-specific promoters, and also have high subcellular specificity through introduction of subcellular targeting sequences, which enables FRET probes to have activity solely in cell types of interest or subcellular regions of interest. QDs have broad absorption spectra and narrow, symmetric emission spectra, resulting in minimal spectral overlap and high FRET efficiency. Dyes and QDs require the assistance of antibodies to label sensing domains effectively, unlike FPs. Small organic dyes are typically composed of conjugated aromatic molecules with a size ranging from a few to several tens of atoms.