Quantitating fluorescence intensity from fluorophores: Practical use of MESF values

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 cytometric measurements. It has been found that emission spectrum matching between a reference solution and an analyte and normalization by the corresponding extinction coefficient 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 are not significantly different, the percentage of error due to spectrum mismatch is estimated. We have also found that the emission spectrum of a microbead with a seven-methylene linker between the fluorescein and the bead surface (bead7) provides the best match with the spectra from biological cells. 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.