Research Flow Cytometry Core

Glossary of Terms

In resonant energy transfer, the molecule that receives the energy transfer and emits a photon.

Raw signal when assay activity is zero.

Intensity of fluorescence generated after excitation of a fluorochrome. The brightness is determined by two optical properties of the fluorochrome: the coefficient of extinction and the quantum yield.

Fraction of light lost to scattering and absorbtion per unit distance in a participating medium. It is the measure of how much light a fluorochrome absorbs.  It is measured in a molar-1 context, i.e. per mole with a defined path length, etc.

The goal of compensation is to remove the spillover fluorescence of a particular fluorophores from the "wrong" channel (secondary fluorochromes). Each detector of a cytometer collects light from multiple sources: principally from the primary fluorochrome, but some light from other fluorochromes as well. The process of compensation is the correction for the light emitted by these secondary fluorochromes.

In resonant energy transfer, the molecule that absorbs a photon and initiates energy transfer to the acceptor.

Energy emitted as photons when excited molecules return to their ground state.

The chemical moiety of a dye molecule which absorbs light and, in the case of a fluorophore, emits light through fluorescence.

Fluorescence Resonance Energy Transfer- In two adjacent dyes, the energy of an excited electron in one dye (the donor) is passed to an electron of an adjacent dye (the acceptor) through resonance, then released as a photon.

Statistical variability in the response of the assay from all sources, including: variation in actual quantities of reagents added or their concentration, instrument output variability, geometry of plates, uncertainty or variability in background, etc.

Upon exposure to light, to decrease in absorbance intensity or, for fluorescent compounds, to decrease in emission intensity.

Two fluorescent dyes selected for their ability to successfully achieve FRET when brought into close proximity in a FRET Assay or in a tandem dye.

Proportion of absorbed photons released as fluorescence.

The process of extinguishing, removing, or diminishing a physical property such as heat or light. Fluorescence quenching, a technique used in investigations dealing with binding of antigens (haptens) by purified antibodies, applicable in cases in which the bound antigen (hapten) absorbs (quenches) light emitted during fluorescence of protein (antibody) excited by ultraviolet light.

Signal before correction for background.

Tendency of fluorophores with small Stokes shifts to absorb photons at some of the same wavelengths at which they emit.

The "real" response of an assay to activity in the sample, independent of all sources of error and statistical variability, corrected for any background.

Crosstalk in fluorescence imaging occurs when the excitation and / or emission spectra of two or more fluorophores (and /or autofluorescence) in a specimen overlap making it difficult to isolate the activity of one fluorophore alone.

Two fluorescent dyes capable of FRET covalently coupled. Tandem dyes are used when increased Stokes shifts or multiple emission wavelengths from a single excitation

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Research Flow Cytometry Core

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