Fluorescence

Fluorescent probes enable researchers to detect particular components of complex biomolecular assemblies, including live cells, with exquisite sensitivity and selectivity. Fluorescence is the result of a three-stage process, see below, that occurs in certain molecules (generally polyaromatic hydrocarbons or heterocycles) called fluorophores or fluorescent dyes. A fluorescent probe is a fluorophore designed to localize within a specific region of a biological specimen or to respond to a specific stimulus.

Stage 1 : Excitation

A photon of energy hv_EX is supplied by an external source such as an incandescent lamp or a laser and absorbed by the fluorophore, creating an excited electronic singlet state (S₁').

Stage 2 : Excited-State Lifetime

The excited state exists for a finite time (typically 1–10 nanoseconds). During this time, the fluorophore undergoes conformational changes and is also subject to a multitude of possible interactions with its molecular environment. These processes have two important consequences. First, the energy of S₁' is partially dissipated, yielding a relaxed singlet excited state (S₁) from which fluorescence emission originates. Second, not all the molecules initially excited by absorption (Stage 1) return to the ground state (S₀) by fluorescence emission. Other processes such as collisional quenching, fluorescence energy transfer and intersystem crossing may also depopulate S₁. The fluorescence quantum yield, which is the ratio of the number of fluorescence photons emitted (Stage 3) to the number of photons absorbed (Stage 1), is a measure of the relative extent to which these processes occur.

Stage 3 : Fluorescence Emission

A photon of energy hv_EM is emitted, returning the fluorophore to its ground state S₀. Due to energy dissipation during the excited-state lifetime, the energy of this photon is lower, and therefore of longer wavelength, than the excitation photon hv_EX. The difference in energy or wavelength represented by (hv_EX –hv_EM) is called the Stokes shift. The Stokes shift is fundamental to the sensitivity of fluorescence techniques because it allows emission photons to be detected against a low background, isolated from excitation photons. In contrast, absorption spectrophotometry requires measurement of transmitted light relative to high incident light levels at the same wavelength.

Fluorescence Spectra

The entire fluorescence process is cyclical. Unless the fluorophore is irreversibly destroyed in the excited state (an important phenomenon known as photobleaching), the same fluorophore can be repeatedly excited and detected. For polyatomic molecules in solution, the discrete electronic transitions represented by hv_EX and hv_EM are replaced by rather broad energy spectra called the fluorescence excitation spectrum and fluorescence emission spectrum, respectively. With few exceptions, the fluorescence excitation spectrum of a single fluorophore species in dilute solution is identical to its absorption spectrum. Under the same conditions, the fluorescence emission spectrum is independent of the excitation wavelength, due to the partial dissipation of excitation energy during the excited-state lifetime. The emission intensity is proportional to the amplitude of the fluorescence excitation spectrum at the excitation wavelength.

Fluorescence Signals

Fluorescence intensity is quantitatively dependent on the same parameters as absorbance — defined by the Beer–Lambert law as the product of the molar extinction coefficient, optical pathlength and solute concentration — as well as on the fluorescence quantum yield of the dye and the excitation source intensity and fluorescence collection efficiency of the instrument. In dilute solutions or suspensions, fluorescence intensity is linearly proportional to these parameters. When sample absorbance exceeds about 0.05 in a 1 cm pathlength, the relationship becomes nonlinear and measurements may be distorted by artifacts such as self-absorption and the inner-filter effect. Because fluorescence quantitation is dependent on the instrument, fluorescent reference standards are essential for calibrating measurements made at different times or using different instrument configurations.

Background Fluorescence

Fluorescence detection sensitivity is severely compromised by background signals, which may originate from endogenous sample constituents (referred to as autofluorescence) or from unbound or nonspecifically bound probes (referred to as reagent background). Detection of autofluorescence can be minimized either by selecting filters that reduce the transmission of E2 relative to E1 or by selecting probes that absorb and emit at longer wavelengths. Although narrowing the fluorescence detection bandwidth increases the resolution of E1 and E2, it also compromises the overall fluorescence intensity detected. Signal distortion caused by autofluorescence of cells, tissues and biological fluids is most readily minimized by using probes that can be excited at >500 nm. Furthermore, at longer wavelengths, light scattering by dense media such as tissues is much reduced, resulting in greater penetration of the excitation light.

Ratiometric Measurements

In some cases the free and ion-bound forms of fluorescent ion indicators have different emission or excitation spectra. With this type of indicator, the ratio of the optical signals (S1 and S2) can be used to monitor the association equilibrium and to calculate ion concentrations. Ratiometric measurements eliminate distortions of data caused by photobleaching and variations in probe loading and retention, as well as by instrumental factors such as illumination stability.