Molecules and atoms can exist in different quantum states. These states are dedicated to different energy levels; the quantum state with the lowest energy is called the ground state. Every state of greater energy is an excited state of the quantum mechanical system. Electrons can be excited by an exogenous energy source and switch to a higher energy level, changing the quantum state of the molecule or atom. The electrons are often referred to as being brought to a state of higher energy.
Molecules whose electrons have been brought to a state of higher energy can emit photons. This process is called luminescence. To reach the excited state, electrons have to be stimulated with a certain amount of energy. It has to match the differences in the energy levels between the two quantum states. This energy can be delivered to the electrons e.g. via the interaction with photons. As photons of different wavelength vary in their energy, the photon needs the appropriate wavelength. Luminescence which is introduced by photons is called photoluminescence.
There are two sub-groups of photoluminescence, fluorescence and phosphorescence. They differ in the quantum state transitions that lead to the luminescence and thereby in the duration of their photon re-emission. Fluorescence ends nanoseconds after the excitation, whereas phosphorescence can last for seconds or in some cases even for hours. Quantum states cannot only be divided into excited and ground states but also into singlet and triplet states which are based on different spin alignments.
The spin is a quantum mechanical property of elementary particles. In simplified terms, it describes the intrinsic angular momentum of a particle, e.g. an electron, caused by its rotation and is characterized via the spin quantum number "s". There are two possible orientations for the spin of an electron, s = +1/2 (positive) or s = -1/2 (negative).
Spin pairs can either be combined parallel (both spins are either positive or negative) or antiparallel (one spin is positive, the other is negative) in their orientation to each other. If the electrons occupy the same atomic orbital, their spins will have to be antiparallel. In antiparallel spin pairs the individual angular momentums compensate each other and the total angular momentum gets a value of zero. This spin alignment is called singlet state. But if two electrons occupy two different orbitals, their spins may be parallel to each other. Two parallel spins do not compensate and get a value different from zero. In this case the spins are said to be in a triplet state.
Fluorescence describes the emission of a photon while an electron transits from an excited singlet state to another singlet state of lower energy, typically the ground state. This transition occurs rapidly after the excitation. Energy is released as a photon. The wavelength of the emitted photon is of greater wavelength than the exciting light, as the emission occurs after the fluorophore has released some of the energy to the surroundings. This phenomenon was discovered by George Gabriel Stokes in 1852 and is named Stokes shift.
Phosphorescence is very similar to fluorescence but it depends on the formation of excited triplet states. Excited triplet states can be formed in some molecules. The paired spins can be uncoupled in a process called intersystem crossing. The intersystem crossing is influenced by the spin-orbit coupling. This involves the interaction of the magnetic moment of an electron due to its spin and the magnetic moment due to its orbital angular momentum.
In the triplet state, the electron releases energy to its surroundings and reaches the triplet ground state. This state is of higher energy than the ground state but also of lower energy than the excited singlet state. The electron cannot switch back to the singlet state and cannot transit to the ground state as the spins are parallel and the transition is now spin-forbidden. But a few changes from the triplet to the ground state are possible at a time. These events of intersystem crossing give rise to the emission of photons. Phosphorescence therefore describes the photon emission during the transition from a triplet state to the ground state.
Other important quantum states for microscopy are dark states. In general, exposure of fluorescent molecules to extremely bright excitation light can lead to the transition of electrons from the excited state to the long-lived dark state via intersystem crossing (ISC). Molecules in the dark state are unable to absorb or emit photons of the original wavelengths for fluorescence and therefore appear “dark”. The triplet state is an example of a dark state. The transition into a dark state is a reversible process.
A process which has to be distinguished from the transition into a dark state is the photobleaching of fluorophores. Photobleaching is an irreversible process that leads to the complete loss of a fluorophore’s ability to fluoresce. The excitation light induces chemical processes that change the molecule and avoid the excitation of the system.
Photobleaching is not fully understood yet. It is likely that bleaching is carried by multiple photon absorption of one electron which is then brought to higher excited states. These higher excited states might give rise to new chemical reactions that might change the structure of a given fluorophore. The temperature and the power of the exciting light influence the bleaching process. With low light power and at low temperatures bleaching is reduced. Attention has to be drawn to potential reactants as well, e.g. oxygen species with their radical character can react with the dye after excitation and lead to bleaching.