Fluorescence intensity is one of the most common measurement techniques in microplate readers. Conceptually it is a simple, a fluorophore is excited at one wavelength and emits light at another wavelength. The emitted light is at a lower energy and thus longer wavelength than the excitation light, and, the amount of emission is directly related to the amount of fluorophore in the sample. The excitation and emission light can be discriminated by filters or monochromators, or, a combination of the two. The emitted fluorescence is detected with a very sensitive photomultiplier tube and dedicated detection circuits that ensure a low amount of noise in the signal.
There are many ways to use fluorescence in life science experiments and there are literally thousands of fluorescent tags available. In the most basic experiments the fluorescence signal will increase as something happens to the analyte of interest, or conversely, the fluorophore emission might be quenched as the environment changes around the analyte. There are fluorophores specific for pH, ionic strength, oxygen content, certain ions and they can even be tied to electric potentials in cells. Fluorescence is one of the most versatile techniques available.
There are advanced fluorescent techniques such as Fluorescent Resonance Energy Transfer, or FRET. FRET is a technique that uses two fluorophores to study binding interactions between molecules. The experiment is setup with a donor and acceptor fluorophore, typically fluorophores where the emission of one overlaps the excitation over another, and when the donor and acceptor are in a binding configuration the fluorescence emission will shift source from the donor wavelength to the acceptor wavelength. By monitoring the ratio of these emissions the interaction between the target molecules can be determined.
Fluorescence intensity detection with BMG LABTECH microplate readers
Fluorescence intensity-based detection can be performed on the PHERAstar® FSX, CLARIOstar®, and FLUOstar® Omega. BMG LABTECH microplate readers use high-intensity xenon flashlamps for excitation with much care given to the high voltage power supplies that drive these lamps. Depending on the reader optical application modules, LVF monochromators™, or filters are used to select the specific wavelengths needed for the application. All BMG LABTECH microplate readers can perform FRET, however, the PHERAstar FSX has a distinct advantage with FRET due to simultaneous dual emission detection where both emission wavelengths are detected at the same time. This PHERAstar FSX only feature doubles speed over all conventional microplate readers, lowers standard deviations, and improves an assay Z’ factor.
The following BMG LABTECH microplate readers can be configured to perform fluorescence measurements: