To visualise or detect substrate binding, it can be advantageous to have an intensity increase. In bulk measurements, this allows bound and unbound species to be identified, whereas, in single-molecule measurements, it allows isolation of protein-bound molecules from background fluorescence.

Intensity changes relate to environmental sensitivity of the fluorescent probe. The quantum yield and extinction coefficient are frequently affected by environmental factors such as pH, solvents and viscosity. Furthermore, they are also affected by structural features of the fluorophore whereby rigid, planar molecules have a higher fluorescence.

As binding sites are frequently more polar than the solvent and in these sites fluorophores can be restricted to planar conformations, positioning of fluorophores in binding sites can allow detection of conformation changes.