spectrofluorimeters

A spectrofluorimeter, or fluorescence spectrophotometer, is an analytical instrument used to measure the intensity and spectral distribution of fluorescence emitted by a sample after it has been excited by light. It provides information about the spectral characteristics of fluorophores and can be used to quantify fluorescent species in solution.

The typical instrument includes a light source (often a Xenon arc lamp or UV LEDs), a sample compartment with a cuvette or microplate, excitation and emission monochromators or filters, and a sensitive detector such as a photomultiplier tube or a cooled solid-state detector. The excitation pathway selects a specific wavelength to illuminate the sample, and the emitted fluorescence is collected and analyzed by the emission pathway. The instrument can operate in scanning mode to acquire emission spectra at a fixed excitation or in excitation mode to obtain spectra by varying excitation wavelength while monitoring emission at a fixed wavelength.

Two common measurement modes are steady-state fluorescence, which records fluorescence intensity across wavelengths, and time-resolved fluorescence, which measures the decay of fluorescence to determine lifetimes. Data are corrected for instrument response and, when necessary, for inner-filter effects or background signals.

Applications include quantitative assays using fluorescent probes, DNA and protein analysis, environmental monitoring of fluorescent contaminants, and studies of binding and conformational changes. Advantages include high sensitivity, broad dynamic range, and non-destructive measurements. Limitations include photobleaching, quenching, spectral overlap, and requirements for suitable fluorophores. Modern instruments may integrate features such as microplate readers, time-correlated single-photon counting, and advanced data processing.