Fluorimetrie

Fluorimetry is a spectroscopic technique that measures the intensity of fluorescent light emitted by a substance after it has absorbed light. The signal depends on the presence and properties of fluorescent species, such as concentration, quantum yield, and environmental factors like solvent, pH, and temperature.

A fluorimeter consists of a light source, an excitation monochromator, a sample holder, an emission monochromator, and a detector. Common light sources include xenon lamps, deuterium lamps, or lasers. The emitted fluorescence is separated from excitation light and detected by a photomultiplier tube or a solid-state detector; data can be collected as emission spectra, excitation spectra, or as signal at a fixed wavelength.

Fluorimetry can be performed in steady-state mode, recording fluorescence intensity under continuous illumination, or in time-resolved mode, measuring fluorescence lifetime after a short excitation pulse. Lifetime measurements, often using TCSPC, help distinguish fluorophores with overlapping spectra and reduce background from autofluorescence.

Quantitative use relies on a linear relationship between fluorescence signal and fluorophore concentration within a working range, while factors such as inner-filter effects, quenching, photobleaching, and matrix interferences must be controlled. Calibration, appropriate dilution, and selection of suitable fluorophores are important.

Applications span biotechnology, clinical diagnostics, environmental monitoring, pharmaceuticals, and food analysis. Fluorimetry is widely used for DNA and protein quantification with fluorescent dyes, detection of labeled biomolecules, and assay development where high sensitivity or multiplexing is advantageous.

In practice, fluorimetry is closely related to fluorescence spectroscopy and is sometimes referred to as spectrofluorimetry or fluorescence spectrometry. It complements absorbance-based methods by offering higher sensitivity for dilute samples.