Fluorescence
Fluorescence is an integral part of optical imaging and can be defined as the process of light emission by molecules that are capable of being excited, via light absorption, from a ground state to an excited state. As shown in the following diagram, fluorophores absorb light energy when exposed to an external light source. The absorbed energy allows the molecules to reach a higher-energy level, or excited state. When the fluorophore falls from the excited to the ground state, the excess energy is released and re-emitted at a longer wavelength. A fluorophore is in a stable configuration and does not fluoresce in the ground state, until it is excited again.
Stokes’ Shift
A fluorescent molecule has two characteristic spectra — excitation and emission. The difference in wavelength, or equivalently, in the energy, between the maxima of the excitation and the emission spectra, which is known as Stokes’ shift, is due to the loss of energy of the excited fluorophore through molecular vibrations. This energy is dissipated as heat before the fluorescent light is emitted.
Brightness and Quantum Yield
Fluorophores differ in their fluorescence yield, which can be defined as the intensity ratio of emitted to absorbed light. Fluorescence yield depends on two properties of the fluorophore — the extinction coefficient and the quantum yield. The extinction coefficient is related to the probability that a photon will be absorbed by a fluorescent molecule, while the quantum yield is the probability that an excited molecule will emit a fluorescence photon. The quantum yield can be affected by such factors as temperature, ionic strength, pH, excitation light intensity and duration, covalent coupling to another molecule, and photo bleaching.
Fluorescence Lifetime
Fluorescence lifetime is the characteristic time that a molecule remains in an excited state prior to returning to its ground state. Typically measured in nanoseconds, fluorescent lifetime is an intrinsic property of the fluorophore that depends on the nature of the fluorescent molecule and its environment. As an added parameter available with time domain technology, fluorescence lifetime measurements can be used to discriminate between fluorophores and to differentiate between probe-derived fluorescence signals and autofluorescence.
Various methods exist to identify and to discriminate signals coming from different fluorophores within the same imaged medium. If the fluorophores have different excitation or emission spectra, it is possible to distinguish them based on those spectral differences. However, if the spectra are too similar, this approach is impracticable. A more versatile approach consists in using time-resolved measurements, such as those obtained by time-correlated single photon counting (TCSPC), that allow discrimination of fluorophores based on their respective fluorescence lifetime, regardless of their respective spectra. The TCSPC data obtained using the Optix system, illustrated below, shows the signals from a fluorophore with a short lifetime (red), a fluorophore with a long lifetime (green), and a mixture of the two (yellow). Although both fluorophores have the same emission spectra, these signals can be separated through lifetime measurements.
