Quantitating fluorescence intensity from fluorophore: The definition of MESF assignment

被引:78
作者
Schwartz, A
Wang, L
Early, E
Gaigalas, A
Zhang, YZ
Marti, GE
Vogt, RF
机构
[1] Ctr Quantitat Cytometry, San Juan, PR 00919 USA
[2] Natl Inst Stand & Technol, Gaithersburg, MD 20899 USA
[3] Mol Probes Inc, Eugene, OR 97402 USA
[4] US FDA, Ctr Biol Evaluat & Res, Bethesda, MD 20892 USA
[5] CDC, Div Sci Lab, Atlanta, GA 30341 USA
关键词
flow cytometer; fluorescein; fluorescence; MESF; microbeads; quantitation; SRM; 1932;
D O I
10.6028/jres.107.009
中图分类号
TH7 [仪器、仪表];
学科分类号
0804 ; 080401 ; 081102 ;
摘要
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.
引用
收藏
页码:83 / 91
页数:9
相关论文
共 13 条
[1]   Technological advances in high-throughput screening [J].
Fernandes, PB .
CURRENT OPINION IN CHEMICAL BIOLOGY, 1998, 2 (05) :597-603
[2]  
Gratama JW, 1998, CYTOMETRY, V33, P166, DOI 10.1002/(SICI)1097-0320(19981001)33:2<166::AID-CYTO11>3.0.CO
[3]  
2-S
[4]  
Hermanson G. T., 1996, BIOCONJUGATE TECHNIQ
[5]  
Klonis N, 1996, J Fluoresc, V6, P147, DOI 10.1007/BF00732054
[6]  
Lakowicz J.R., 2004, PRINCIPLES FLUORESCE, VSecond
[7]   Reversible photobleaching of fluorescein conjugates in air-saturated viscous solutions: Singlet and triplet state quenching by tryptophan [J].
Periasamy, N ;
Bicknese, S ;
Verkman, AS .
PHOTOCHEMISTRY AND PHOTOBIOLOGY, 1996, 63 (03) :265-271
[8]  
Schwartz A, 1996, CYTOMETRY, V26, P22, DOI 10.1002/(SICI)1097-0320(19960315)26:1<22::AID-CYTO4>3.0.CO
[9]  
2-I
[10]  
Sjöback R, 1998, BIOPOLYMERS, V46, P445