4.8. Appendix

4.8.1. Fluorescence, phosphorescence and chemiluminescence

Even though the phenomena mentioned in the title are similar in many ways, it is important to make distinctions. In all three cases, the source of the light is an excited electron that, while returning to its ground state, can emit part of its excitation energy as a photon with some probability.

As mentioned earlier, in the case of fluorescence, excitation is performed using light of a wavelength that is optimally absorbed by the molecule. The transition between the excited and ground states is direct and fast (occurs on the nanosecond time scale).

In the case of phosphorescence, the difference lies in the manner and rate of emission. In this case, the electron does not „immediately” fall back from its excited state into its ground state, but is able to enter a particular alternative excited state. All transitions leading from this state to the ground state are so-called forbidden transitions. This does not mean that they do not happen at all, but their probability is rather low. This way, the lifetime of the excited state can increase from nanoseconds to milliseconds, minutes or even hours.

The difference between fluorescence and chemiluminescence is not in the way the emission occurs, but in how the excitation is achieved. In the latter case the energy necessary to excite the electron comes not from light but from a chemical reaction. This is the way some living organisms can produce light. We call this bioluminescence. Fireflies and anglerfishes of deep seas are well known examples of the occurrence of this phenomenon.

4.8.2. Photobleaching

Besides the effects listed above, excitation of fluorophores can also destroy them. This phenomenon is known as photobleaching. Photobleaching can occur during every excitation event with a given probability. Thus, its extent will be proportional to the intensity of the exciting light and the duration of the illumination. This effect can pose a serious problem when conducting long-term experiments. To counteract photobleaching, more durable fluorophores have been invented (although the rate of decay is not zero even in the case of these molecules).

Photobleaching can also be used to our advantage, as exemplified by the FRAP (Fluorescence Recovery After Photobleaching) technique. Using FRAP we can examine e.g. the movement of membrane-associated molecules in cells. After fluorescent labelling a molecule of interest in the membrane, we can induce the decay of fluorophores purposefully in the vicinity of the membrane. Subsequently we can assess how rapidly the fluorophores in the surrounding area enter the bleached patch.

4.8.3. Fluorescence anisotropy and circular dichroism

The properties of light are not completely characterised by its intensity and wavelength. It also has polarisation, which is the function of the direction and the fluctuation of the electric field vector. (All photons are polarised; unpolarised light is the mixture of differently polarised photons). Polarisation can be linear (when the electric field vector changes in a given plane) or circular (when it changes in circles) (Figure 4.17). Both types of polarisation can be utilised in spectroscopy.

Linear and circular polarisation of light

Figure 4.17. Linear and circular polarisation of light. The figure shows an electromagnetic wave (light), a transversal wave propagating vertically and the curve we get by following the electric field vector (blue line) creating it. It also shows what we get if we split the vector into its perpendicular components (red and green). Source: http://scienceblogs.de/astrodicticum-simplex/wp-content/blogs.dir/28/files/2012/06/i-897a5d594e065d9046b61bb92a8d3d02-polarisation.png

Linear polarisation has an effect on fluorescence through the efficiency of the excitation. In addition, the polarisation of the emitted light is a function of the position of the fluorophore. This phenomenon is known as fluorescence anisotropy. Using this effect, we can examine the rotation of a fluorophore, which can provide information on the size and environment of the labelled molecule (larger molecules or those located in more tightly packed environments will rotate slower). If the labelled molecule binds to something of a significant size (e.g. a protein), this event will appear in the experiment as a change in the size of the label-containing particle. This way, association/dissociation reactions can be studied.

Utilising circular polarisation is one of the possible ways to examine the chirality of molecules. Circularly polarised light is absorbed differently by the two forms of chiral molecules (e.g. L and D amino acids). This difference is called circular dichroism and is usually described by the difference between the extinction coefficients of the two enantiomers (Δε). Plotting this parameter against the wavelength we get a so-called circular dichroism (CD) spectrum. It has been observed that polymers with a non-bilaterally symmetric structure also exhibit dichroism. Thus, via CD measurements performed in the UV range, the ratio of the different secondary structures in proteins and the double helix in nucleic acids can be studied. Although this measurement does not provide enough information to determine the exact structure of molecules, we can gain important insights into their general features and detect structural changes. Consequently, circular dichroism measurements can be used to observe the folding and denaturation of proteins. It has also been shown that metal ions show CD in the visible spectrum when they are located in a chiral environment. As they lose this attribute when they are in solution, this effect can be utilised to assess the formation of metalloprotein complexes.

4.8.4. Quenching and FRET

Attentive readers may have noticed that we always described fluorescence emission as one of multiple possible ways to lose the energy of excitation. There are multiple reasons for this:

  1. The entirety of the energy can be dissipated as heat.

  2. Some electrons can enter a third, alternative state (cf. phosphorescence).

  3. It is possible to transfer the energy to a nearby molecule called acceptor.

Phenomena in the last category are called quenching. If the acceptor molecule is not fluorescent, the transfer of energy will lead to loss of fluorescence. If the acceptor is fluorescent, by exciting the donor we will be able to detect acceptor emission, with the consideration of certain spectral requirements.

Quenching can occur through different mechanisms, of which the most common is FRET (Förster Resonance Energy Transfer). The transfer of energy happens in a non-radiative manner, i.e. there is no photon emission by the donor molecule. For this to happen, two prerequisites must hold: compatibility and physical proximity of the molecules involved. The emission spectrum of the donor and the excitation spectrum of the acceptor must overlap with each other. The efficiency of FRET is inversely proportional to the sixth power of the distance between the donor and the acceptor. Generally, if they are located farther apart than 10 nm, there is practically no interaction. This makes FRET a suitable effect for methods to study the distance and interaction of molecules or, with appropriate labelling, even parts of molecules.

In the basic approach, the distinction between the “near” and “far” states is made by observing the emission of the acceptor, based on its wavelength. This requires the donor and acceptor molecules to be different. But even if the two are identical, there is a possibility to detect FRET using an anisotropy measurement, as the latter also undergoes changes upon energy transfer.