Fluorescence Microplate Readers
Fluorescence intensity detection is a technique widely used in life science research, representing the most popular detection mode for microplate readers. Detecting fluorescence requires sophisticated instrumentation that contains an optical system consisting of two building blocks. First, a sample gets illuminated by a light source at a specific wavelength in the excitation part of the optical system. The specific wavelength is typically selected by an optical filter or a monochromator. This results in fluorescence emitted by the sample. The optical pathway of the second part of the optical system (typically a filter or a monochromator system) collects the emitted light and measures its intensity on a light detecting device, e.g. a photomultiplier tube (PMT).
Fluorescence Resonance Energy Transfer (FRET) is a physical phenomenon, that requires close proximity of a donor molecule and an acceptor molecule (1-10 nm typically). As a result of excitation by light that only the donor can absorb, the donor dye changes into an excited state and transfers energy without the emission of a photon to the acceptor dye. If the acceptor dye is fluorescent, it will emit light, typically at a much longer wavelength than the donor dye can emit itself. Today, FRET techniques are getting employed in a wide variety of applications, such as receptor/ligand studies, membrane fusion assays, fluorogenic protease assays or immunoassays.