Fluorometrien

Fluo­rometrien, commonly called fluorometry, is an analytical technique that measures the intensity of fluorescence emitted by a sample after excitation by light. A fluorophore absorbs photons at a shorter excitation wavelength and emits at a longer emission wavelength, with the emitted intensity often proportional to fluorophore concentration. Factors such as quantum yield, extinction coefficient, and photostability influence signal strength and measurement reliability.

A fluorometer typically includes a light source (such as LEDs, a xenon lamp, or a laser), an excitation monochromator, a sample compartment, an emission detector and an emission monochromator or set of filters. The detector is usually a photomultiplier tube or a sensitive photodiode. Many instruments offer time-resolved measurements to determine fluorescence lifetimes, which can separate signals from autofluorescence and reduce background. It is important to correct for stray light and inner-filter effects when quantitative results are required.

Steady-state fluorometry records emission intensity at a chosen wavelength while varying excitation or analyte concentration. Time-resolved fluorometry measures how quickly fluorescence decays after a brief pulse, yielding lifetimes that can discriminate overlapping spectra and provide kinetic information.

Applications include quantitative assays of nucleic acids and proteins with fluorescent dyes, enzyme kinetics, cell biology assays, and environmental monitoring with fluorescent probes. Endogenous fluorophores (for example, NADH and FAD) enable label-free studies in living systems. Advantages of fluorometry include high sensitivity and a wide dynamic range; limitations include photobleaching, autofluorescence from complex samples, spectral overlap, and the inner-filter effect requiring careful calibration.