title: Quantitating Fluorescence Intensity from Fluorophore: The Definition of MESF Assignment.
abstract: The quantitation of fluorescence radiance may at first suggest the need to obtain the number of fluorophore that are responsible for the measured fluorescence radiance. This goal is beset by many difficulties since the fluorescence radiance depends on three parameters 1) the probability of absorbing a photon (molar extinction), 2) the number of fluorophores, and 3) the probability of radiative decay of the excited state (quantum yield). If we use the same fluorophore in the reference solution and the analyte then, to a good approximation, the molar extinction drops out from the comparison of fluorescence radiance and we are left with the comparison of fluorescence yield which is defined as the product of fluorophore concentration and the molecular quantum yield. The equality of fluorescence yields from two solutions leads to the notion of equivalent number of fluorophores in the two solutions that is the basis for assignment of MESF (Molecules of Equivalent Soluble Fluorophore) values. We discuss how MESF values are assigned to labeled microbeads and by extension to labeled antibodies, and how these assignments can lead to the estimate of the number of bound antibodies in flow cytometer measurements.
authors.affiliation: Center for Quantitative Cytometry, PO Box 194344, San Juan, Puerto Rico 00919.
authors.forename: Abe
authors.initials: A
authors.lastname: Schwartz
authors.affiliation: National Institute of Standards and Technology, Gaithersburg, MD 20899-8312.
authors.forename: Lili
authors.initials: L
authors.lastname: Wang
authors.affiliation: National Institute of Standards and Technology, Gaithersburg, MD 20899-8312.
authors.forename: Edward
authors.initials: E
authors.lastname: Early
authors.affiliation: National Institute of Standards and Technology, Gaithersburg, MD 20899-8312.
authors.forename: Adolfas
authors.initials: A
authors.lastname: Gaigalas
authors.affiliation: Molecular Probes, Inc., PO Box 22010, Eugene, OR 97402-0469.
authors.forename: Yu-Zhong
authors.initials: YZ
authors.lastname: Zhang
authors.affiliation: Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, NIH Building 29B, Room 2NN08, Bethesda, MD 20892.
authors.forename: Gerald E
authors.initials: GE
authors.lastname: Marti
authors.affiliation: Division of Laboratory Sciences, CDC, Mailstop F19, 4770 Buford Highway, Atlanta, GA 30341.
authors.forename: Robert F
authors.initials: RF
authors.lastname: Vogt
keywords: MESF; SRM 1932; flow cytometer; fluorescein; fluorescence; microbeads; quantitation