Fluorescence

Fluorescence is a luminescent process in which a substance absorbs light at one wavelength and then emits light at a longer wavelength. It occurs when a molecule absorbs a photon, promoting an electron from the ground state to a singlet excited state. The molecule relaxes through rapid vibrational cooling to the lowest vibrational level of the excited state before emitting a photon and returning to the ground state. Emission typically occurs within nanoseconds and is characterized by a Stokes shift, the difference between excitation and emission wavelengths.

Mechanistically, fluorescence involves radiative decay from the first excited singlet state to the ground state. Nonradiative pathways, such as vibrational relaxation and internal conversion, compete with emission. Environment can affect intensity and wavelength. In some cases, molecules undergo intersystem crossing to a triplet state, leading to phosphorescence or delayed fluorescence. The efficiency is described by the quantum yield, and the lifetime by the fluorescence lifetime.

Fluorescence is distinct from phosphorescence, which arises from triplet-state decays and usually has longer lifetimes. Fluorescence is generally prompt and requires available singlet states; phosphorescence often persists after excitation is removed and is sensitive to temperature and quenching.

Fluorophores include natural substances (chlorophyll, NADH) and synthetic dyes (fluorescein, rhodamine), as well as fluorescent proteins like GFP. Applications span spectroscopy, microscopy, flow cytometry, and in vivo imaging. Fluorescent probes enable detection and quantification of biological molecules, cellular processes, and chemical environments. Quantum dots and other nanomaterials expand fluorescence to greater brightness and stability.

Measurement often records emission spectra while exciting at specific wavelengths, and assesses quantum yield and lifetimes. Fluorescence is sensitive to environment, pH, polarity, and quenchers such as oxygen, making it useful for sensing but requiring careful controls to avoid artifacts.