There are certain principal and practical limitations for fluorescence microscopy. Compared with the power of the excitation light, the observable fluorescence is a relatively weak phenomenon. The ratio of fluorescence and excitation typically ranges between 10-4 and 10-6, but in certain techniques (fluorescence in-situ hybridization etc.) it can be down to
10-9 or 10-10. This has obvious direct consequences for the system requirements. Because intensity is directly correlated to the amount of fluorescent molecules within a certain volume, the preparation of the sample and the efficiency of labelling (staining, expression) is a critical factor.
Critical parameters of the dye are its extinction coefficient, which describes the efficiency to absorb excitation light and furthermore its ability to re-radiate the absorbed photons as fluorescence. Both factors influence the quantum efficiency (quantum yield), which is simply the ratio of absorbed and emitted photons.
An additional parameter of the dye is photobleaching, which describes the chemical destruction of the dye that goes along with the irradiation. An environmental factor is the so-called quenching, which is the radiationless deactivation of excited dye molecules.