fluorophoresfluorescent

Fluorophores are chemical entities that can absorb light at one wavelength and re-emit it at a longer wavelength, a phenomenon known as fluorescence. The absorbed energy excites electrons to a higher singlet state; when they return to the ground state, photons are emitted. The emitted light is typically red-shifted relative to the excitation light, a property called the Stokes shift. Fluorescence is widely used in biological and chemical analyses because emitted light can be detected with high sensitivity against low autofluorescence.

Key properties of fluorophores include their excitation and emission spectra, extinction coefficient, quantum yield, brightness, and photostability. Brightness is often approximated by the product of the extinction coefficient and the quantum yield. Photobleaching refers to the loss of fluorescence under illumination, a common limitation in long experiments. Some fluorophores are environment-sensitive and change spectra with pH, ion concentration, or binding state, which can be exploited as probes.

Fluorophores come in several classes, including organic dyes (for example fluorescein and rhodamine variants and Alexa dyes), fluorescent proteins (such as GFP and mCherry), and inorganic probes like quantum dots. They are used in fluorescence microscopy, flow cytometry, fluorescence in situ hybridization, DNA sequencing, and biosensing. Fluorescence resonance energy transfer (FRET) uses pairs of fluorophores to study molecular interactions.

Labeling strategies include covalent dye tagging, antibody conjugates, and genetic tagging with fluorescent proteins. Practical considerations include spectral planning to avoid overlap, autofluorescence minimization, photostability management, and compatibility with multiplexing. Ongoing developments aim to improve brightness, stability, and emission in the near-infrared, while reducing toxicity and background signals.