Quantitating fluorescence intensity from fluorophore: Assignment of MESF values

被引:35
作者
Gaigalas, AK [1 ]
Wang, LL
Schwartz, A
Marti, GE
Vogt, RF
机构
[1] Natl Inst Stand & Technol, Gaithersburg, MD 20899 USA
[2] Ctr Quantitat Cytometry, San Juan, PR 00919 USA
[3] US FDA, Ctr Biol Evaluat & Res, Bethesda, MD 20892 USA
[4] CDC, Div Sci Lab, Atlanta, GA 30341 USA
关键词
cytometry; FITC; fluorescein; fluorescence yield; MESF; microspheres;
D O I
10.6028/jres.110.010
中图分类号
TH7 [仪器、仪表];
学科分类号
0804 ; 080401 ; 081102 ;
摘要
A procedure is presented to convert the comparison of measured fluorescence signals into a comparison of fluorescence yields (FY). The fluorescence yield, which is a property of a solution or a suspension, is defined as the product of the fluorophore concentration and the molecular quantum yield. The paper revises the measurement model which relates the measured fluorescence signal to the FY. The equality of FY of two solutions provides an equivalence between the concentrations of fluorophore in the two solutions. The equivalence is the basis for quantitation in terms of molecules of equivalent soluble fluorophore (MESF). The quantitation procedure starts with the measurement of fluorescence signals from a serial dilution of fluorescein solutions to obtain a calibration of a fluorometer. The fluorometer is used to measure the fluorescence signal of a suspension of microspheres with immobilized fluorescein isothiocyanate (FITC). The calibration is used to obtain the concentration of soluble fluorophores which gives the same fluorescence signal as the microsphere suspension. The number concentration of microspheres is measured and the equality of fluorescence yields is used to obtain the number of soluble fluorescein molecules equivalent to a single microsphere.
引用
收藏
页码:101 / 114
页数:14
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