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.

Keywords: MESF; SRM 1932; flow cytometer; fluorescein; fluorescence; microbeads; quantitation.

Fig. 1

A schematic diagram of the apparatus used to measure the fluorescence intensity from a solution (suspension) in a cuvette or a flow thru cell. The laser beam is coming out of the plane of the figure and is represented by the dot in the cuvette. The pair of lenses (L) focus the illuminated region in the cuvette on the monochromator entrance slit (M=1). The holographic notch filters (H) are tuned to reject the laser radiation wavelength, in this case 488 nm. The entrance slit is imaged and dispersed on the CCD detector.

Fig. 2

The measured relative absorption and relative fluorescence emission of fluorescein in a pH 9 borate buffer. The emission spectrum was taken with the apparatus shown in Fig. 1, and the excitation spectrum was taken with a SLM 8000 spectrofluorimeter. The relative emission function S(λ) was obtained by dividing the detector response by the response at 515 nm. The integral of the function S(λ) shown in the figure is not normalized to 1.

Fig. 3

A graphical display of the equivalence of fluorescence yields of a microbead suspension and reference fluorophore solution. The straight line is a fit to the six points representing the measured fluorescence radiance in six dilutions of the reference solution. The horizontal arrow points to the measured fluorescence yield of the microbead suspension, and the vertical arrow points to the equivalent concentration of soluble fluorophores.

Fig. 4

A schematic diagram of the geometry that is used to convert a volume fluorescence source into a equivalent surface source. A_B is the cross sectional area of the illuminating laser beam which is coming out of the plane of the figure. The source plane intersects the illuminating beam at its mid section. A_E is the area of the entrance aperture of the collecting optics, and D is the distance between the entrance aperture and the source plane. Finally, A_S is the area of the image of the monochromator entrance slit on the source plane. (A_S < A_B). The refraction at the cuvette wall is not shown in the figure. A radiance standard would replace the cuvette during calibration. The surface of the radiance standard is placed at the source plane.