What is fluorescence?

Fluorescence can be defined as the absorption of electromagnetic radiation, typically from ultraviolet or visible light, by an atom or molecule and the subsequent emission of photons of lower energy. In fluorescence, the emission lasts a very short time: in the order of nanoseconds to microseconds after radiation has been absorbed; if the emission lasts much longer (in the order of milliseconds or even many seconds), the phenomenon is called phosphorescence. Fluorescence and phosphorescence are the main types of photoluminescence, one of the subtypes of luminescence.

Jablonski diagram. Picture by Jacobhked: https://commons.wikimedia.org/wiki/User:Jacobkhed

Mechanism of fluorescence

The absorption of electromagnetic radiation (typically in the UV or visible spectrum) moves electrons from their ground state (called S0) to energetically higher levels (S1, S2, S3…) in a process called photoexcitation. Depending on the energy of the photon, this could correspond to a change in vibrational, electronic, or rotational energy levels; the changes between these levels are called transitions. After the excitation, electrons can return to a lower energy level through various relaxation processes: in the case of fluorescence, electrons lose energy by radiative transition, that is, by emitting photons, and in all transitions, electrons stay in singlet state. The main transitions are plotted on the Jablonski diagram (see picture).

In some circumstances, excitation can happen through nonradiative dipole–dipole coupling instead of normal absorption of photons. This is the case of FRET (Förster Resonance Energy Transfer of Fluorescence Resonance Energy Transfer), in which a donor fluorophore excites an acceptor fluorophore nonradiatively when both are in close proximity (typically 1-10 nm).  

Fluorescence spectra of rhodamine 6G showing the Stokes shift. By Sobarwiki - Own work, Public Domain, https://commons.wikimedia.org/w/index.php?curid=29474504

Characteristics of fluorescence

There are some characteristics of fluorescence that are relevant for their applications:

  1. Stokes shift: it’s the shift of the emission peak respective to the excitation peak. As excitation light can interfere with the measurement of emission light, for most applications it is beneficial to use fluorophores with a large stokes shift.
  2. Lifetime: the fluorescence lifetime refers to the average time the molecule stays in its excited state before emitting a photon, and it is characteristic of the fluorophore and its environment. Fluorescence lifetime has important implications for their applications: for example, fluorophores with a very long lifetime can be used to remove interferences from other fluorescent substances in the sample using TRF (time-resolved fluorescence), and some molecular interactions can reduce the lifetime of the fluorescence, which can hence be used to detect such interactions.

Advantages of fluorescence

Fluorescent molecules can be detected by the light they produce, and the emission happens at specific wavelengths, and only when excited at specific wavelengths. This makes fluorescence ideal to detect and quantify molecules. Quantification can be performed either directly, if the substance to be determined is fluorescent itself, or using a molecule labelled with fluorescence that binds to the molecule of interest, such as an antibody or probe, or by means of an intermediate reaction that generates, quenches or releases a fluorescent product.

Compared to methods based on absorbance, fluorescence-based methods offer higher sensitivity (typically 10-fold).

The amount of different fluorescent proteins and the broad range of spectral properties offers great flexibility and is particularly suited for multiplex assays, that is, assays determining multiple parameters from the same sample. Fluorescent molecules are characterized not only by its excitation and emission spectra, but also for other properties, such as fluorescence lifetime and polarization. Those properties can also be used in some methods, further increasing the versatility of fluorescence-based methods.

Limitations of fluorescence

In most cases the molecule of interest is not fluorescent itself. This means that it has to interact with some fluorescent substances before it can be quantified. Strategies to achieve this include using antibodies or nucleic acid probes that are labelled with fluorescence, using intercalating dyes (in the case of nucleic acids), generating fusion proteins with fluorescent properties, chemical reactions that generate or destroy a fluorescent substance, and others.

Biological samples often contain fluorescent molecules, which can interfere with the detection of the molecule of interest.

Most fluorophores suffer from photobleaching, that is, a decrease in fluorescence (due to the destruction of the fluorophore) as a result of its exposure to excitation light. This limits the time (or number of times) the sample can be measured: the longer the sample is excited, the lower the fluorescence will be. While this is normally not a problem for endpoint measurements, it can compromise the results in the case of long kinetic measurements or microscopy applications.

Compared to luminescence-based methods, fluorescent assays are less sensitive (typically 10- to 100-fold).