Fluorometry

Fluorometry is a spectroscopic technique that measures the fluorescence emitted by a sample after it absorbs light of a shorter wavelength. When a fluorophore absorbs photons, it is promoted to an excited electronic state and, after non-radiative relaxation, returns to the ground state by emitting a photon with a longer wavelength. The result is a spectrum of emitted light (emission spectrum) that is typically measured while scanning the emission wavelength, often with a fixed or variable excitation wavelength (excitation spectrum). The difference in wavelengths between excitation and emission is called the Stokes shift.

Instrumentation and operation involve a fluorometer or spectrofluorometer consisting of a light source, an excitation monochromator, a sample compartment, an emission monochromator, and a detector such as a photomultiplier tube. Measurements can be steady-state, recording fluorescence at a constant excitation and emission condition, or time-resolved, which uses pulsed light to determine fluorescence lifetimes.

Fluorometry yields data such as fluorescence intensity, spectra, quantum yield (the fraction of excited molecules that emit photons), and, in time-resolved mode, fluorescence lifetimes. Fluorophores can be intrinsic (such as tryptophan in proteins) or externally labeled with fluorophores.

Applications span quantitative assays (protein, nucleic acid, and enzyme assays), environmental monitoring, clinical diagnostics, and fluorescence imaging. Advantages include high sensitivity and selectivity when labeling is used, while limitations involve photobleaching, inner-filter and background autofluorescence, and potential quenching or turbidity effects. Proper calibration, blanks, and spectral corrections are essential for accurate measurements.