These properties are all related to the excitation and emission cycle of the fluorophore (Fig. 1). A fluorophore is excited to high energy singlet states (S1 and/or S2) when it absorbs a photon. From the excited state, the molecule must relax to the lowest energy level of S1 through a non-radiative loss of energy. From this state the fluorophore can release a photon as fluorescence to return to the ground state. The molecule can also cross from the singlet state to the triplet state (T1) through intersystem crossing. Relaxation from T1 back to the ground state occurs through non-fluorescent processes or phosphorescence.
Fig. 1 Excitation and emission of a fluorophore. Absorption of light at a particular wavelength causes the fluorophore to move from a ground state (S0) and become excited in a higher electronic energy level (either S1 or S2). Different vibrational levels exist in each electronic energy level. Following this excitation, the fluorophore relaxes to the lowest vibrational state of S1 through internal conversion in ~ 10-10 sec. Following internal conversion, the fluorophore remains in the S1 energy state for a defined amount of time, termed the fluorescence lifetimes (10-9 sec). The fluorophore returns to the ground state through release of a photon at a particular wavelength in the process of fluorescence.
A fluorophore’s brightness is defined by two parameters, its extinction coefficient and its quantum yield. The extinction coefficient is a measure of the ease in which a fluorophore absorbs light to enter the excited state. Once a photon has been absorbed, the probability of a photon being emitted through fluorescence is related to the quantum yield. Therefore, a molecule with a high quantum yield and extinction coefficient is the brightest.
The fluorescent lifetime is the average time the molecule spends in the excited state. The lifetime is dependent upon the fluorophore’s environment and corresponding changes in lifetime correlate with intensity changes. As will be described below, the lifetime is an important parameter for fluorescence polarization/anisotropy measurements.
During the relaxation in S1, the loss of energy due to non-radiative decay, before the release of a photon, leads to an emission maximum at a lower energy (higher wavelength) than the excitation. The difference between the excitation and emission wavelength is the Stoke’s shift, and the degree of shift is related to the molecular structure of the fluorophore.
Fluorophores will go through many cycles of excitation and emission but all will eventually be irreversibly deactivated, or photobleached. During these cycles, approximately 1:1000 excitations leads to a transition from the singlet state (S1/S2) to the triplet state (T1). This state is long-lived (millisecond) and the fluorophore will not emit again until it reaches the ground state and is re-excited. These “dark” periods give rise to the phenomena of fluorophore blinking. It is during the long-lived triplet state that the fluorophore can react with oxygen and free radicals leading to irreversible deactivation. Established methods exist to remove oxygen from solution using scavenger enzymes such as glucose-oxidase and catalase . Triplet state quenchers, such as trolox, and redox systems, such as methyviologen and ascorbic acid, can further reduce the photobleaching rate by quenching the highly reactive state.
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Toseland, C.P. “Fluorescent labelling and modification of Proteins”. J. Chem. Biol. 2013 6:85-95.