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Quantitating Fluorescence Intensity From Fluorophores: Practical Use of MESF Values

Published

Author(s)

Lili Wang, Adolfas K. Gaigalas, F Abassi, G E. Marti, R F. Vogt, A Schwarz

Abstract

The present work uses fluorescein as the model fluorophore and points out critical steps in the use of MESF (Molecules of Equivalent Soluble Fluorophores) values for quantitative flow cytomeric measurements. It has been found that the emission spectrum matching between a reference solution and an analyte and normalization by the corresponding extinction coeffecient are required for quantifying fluorescence signals using flow cytometers. Because of the use of fluorescein the pH value of the medium is also critical for accurate MESF assignments. Given that the emission spectrum shapes of microbead suspensions and stained biological cells do not vary significantly, the percentage of error due to spectrum mismatch is estimated using the simple research flow cytometer as the model cytometer. We have also found that the emission spectrum of the microbead with seven-methylene linker between fluorescein and bead surface (bead7) matches those of biological cells the best. Therefore, bead7 is potentially a better calibration standard for flow cytometers than the existing one that is commercially available and used in the present study.
Citation
Journal of Research (NIST JRES) -
Volume
107
Issue
4

Keywords

emission spectrum matching, extinction coefficient, fluorescein, lymphocyte, MESF value, microbead, pH, quantitative flow cytometry

Citation

Wang, L. , Gaigalas, A. , Abassi, F. , Marti, G. , Vogt, R. and Schwarz, A. (2002), Quantitating Fluorescence Intensity From Fluorophores: Practical Use of MESF Values, Journal of Research (NIST JRES), National Institute of Standards and Technology, Gaithersburg, MD (Accessed February 22, 2024)
Created July 1, 2002, Updated February 17, 2017