Fluorescence intensity detection is the measurement of the light emitted by a fluorescent molecule (fluorophore) upon excitation by light at a higher energy and smaller wavelength. A sample is excited by the light produced by a light source and filtered at a specific wavelength by either a filter or a monochromator. The excited sample emits light usually at a lower energy and longer wavelength than the excitation light and fluoresces within µseconds upon excitation. The emitted light is filtered, collected, and measured by a detector, usually a Photo Multiplier Tube (PMT), and quantified in Relative Fluorescence Units (RFU). The vast development of countless fluorophores over the last two decades has made fluorescence intensity one of the most popular detection modes for microplate readers, with thousands of assays available for many different applications.
FRET is a dual dye fluorescence detection assay based upon Fluorescence Resonance Energy Transfer between a donor and an acceptor. If donor and acceptor are in close proximity (10-100 Å), excitation of the donor fluorophore transfers energy to the acceptor fluorophore that will emit light without being directly excited. FRET is mainly used to monitor interactions between molecules.
Fluorescence intensity detection with BMG LABTECH microplate readers
Fluorescence intensity-based detection can be performed on the PHERAstar® FSX, CLARIOstar®, and SPECTROstar® Omega. A high-intensity xenon flashlamp in all BMG LABTECH instruments provides high performance measurements, while assay-specific filters, optic modules, or LVF monochromators™ select the specific wavelengths, yield higher light transmission, and minimize unwanted background noise. Low-noise or extended red-shifted PMTs provide, combined with a user-adjustable gain, a measurement window of more than 6 logs intensity.
The following BMG LABTECH microplate readers can be configured to perform fluorescence measurements: