Quantitating fluorescence intensity from fluorophores: Practical use of MESF values

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 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.

The development of quantum dot calibration beads and quantitative multicolor bioassays in flow cytometry and microscopy

“…Running a sample of commercially available standard calibration beads in flow cytometry experiments allows normalization of multiple data sets, even if acquired with different detector settings or on different instruments (9,10). The utility of calibration beads lies in the application of the concept of MESF (9,10). MESF values rely on the equivalency of fluorescence intensity between a suspension of fluorophore bearing-beads and soluble fluorophores of the same species.…”

“…The close agreement between independent semi-theoretical expectations (Equations 3 and 4) can be viewed as a performance validation of our quantum dot calibration beads measured against the industry yardstick Quantum ™ FITC MESF beads. It is useful to note that workers at the National Institute of Standards and Technology (NIST) have published a detailed characterization of the applicability of the MESF beads in quantitative flow cytometry (9,10). A potential source of systematic error, which we have not seriously considered here, is the error introduced by the dichroic filter as a result of spectral mismatch between the emission spectrum of the sample (fluorescein biotin) and the spectrum of the standard calibration beads or our QD525 beads.…”

“…A potential source of systematic error, which we have not seriously considered here, is the error introduced by the dichroic filter as a result of spectral mismatch between the emission spectrum of the sample (fluorescein biotin) and the spectrum of the standard calibration beads or our QD525 beads. This error can be as high as 18% for a spectral shift of up to 15nm, and it arises from changes in the transmission efficiency of the dichroic filter in a model flow cytometer in the 515-545 wavelength range delimited by the 530 bandpass filter (9,10). It is also instructive to note that the quantum yields of some fluorophores (e.g.…”

“…The use of standard calibration beads to define the quantity of fluorophore-tagged ligands or proteins on cells or beads represents an essential element of quantitative flow cytometry, that has enabled the direct comparison of inter-laboratory data as well as quality control in clinical flow cytometry (9)(10)(11)(12). The utility of these standard beads is based on the correlation between the fluorescence intensity of a solution of known concentration and the intensity of a suspension of beads bearing the same fluorophores of indeterminate surface density.…”

“…The utility of these standard beads is based on the correlation between the fluorescence intensity of a solution of known concentration and the intensity of a suspension of beads bearing the same fluorophores of indeterminate surface density. The equivalence of fluorescence radiance of the beads to the solution is known as the Molecules of Equivalent Soluble Fluorophores (MESF) where the MESF value is equal to the known number of molecules in solution (9,10). To the best of our knowledge the only commercially available sets of calibration beads are based on fluorescein, cyanine-5 (cy5) and phycoerythrin (PE) available from BangsLabs, Fiskers, IN) and EGFP from BD Biosciences.…”