Fluorescence intensity (incl. FRET)
Fluorescence is the emission of light by a molecule (called fluorophore) that has been excited by light with a shorter wavelength than the emitted one. Fluorescence intensity detection is the measurement of this emitted light. A fluorescence plate reader consists of a light source, an excitation filter or monochromator, an emission filter or monochromator and a detector, usually a photomultiplier tube (PMT). As a result of a wavelength-specific excitation (light selected by the excitation filter/monochromator), the fluorophore emits light that is separated from the excitation light by the emission filter/monochromator and is measured by the PMT.
Fluorescence intensity is not an absolute measurement and is usually quantified in Relative Fluorescence Units (RFU). Thanks to the vast development of different fluorophores over the last decades, fluorescence intensity has become one of the most popular detection modes for plate readers, with thousands of assays available for many different applications.
Common applications for fluorescence intensity are calcium flux, DNA quantification, gene expression, protein interaction via Fluorescence Resonance Energy Transfer (FRET), enzymatic activities and more.
- Cell-based assay detects residual β-blocker substances in effluent of municipal wastewater treatment plants
- FRET sensor assesses ATP levels in living plants experiencing low oxygen conditions
- Calcium retention capacity assay evaluates inhibition of mitochondrial permeability transition pore
- Protein-induced fluorescence enhancement detects protein-nucleic acid interactions in microplates
- Real-time assessment of apoptosis and necrosis