Resolution of dimly fluorescent particles: a practical measure of fluorescence sensitivity

Flow cytometry is usually used to analyze subpopulations of cells, not simply to measure the mean fluorescence level of a mixture. Thus, resolution or coefficient of variation (CV) of dimly stained populations is the most appropriate measure of fluorescence "sensitivity." Methods used to measure sensitivity that are in routine use do not unambiguously and completely determine the ability of a flow cytometer to resolve dimly fluorescent populations from each other. Since fluorescence sensitivity depends on two factors, background light (B) and detection efficiency (Q, the detected photoelectrons per fluorochrome molecule on an analyzed particle), one cannot uniquely define the operating condition of a flow cytometer with just one of these factors. In general, it is not possible to define the ability of a flow cytometer to resolve dim subpopulations by using a single number such as "noise level" or "detection threshold"-the description requires a "two-parameter" measure. A carefully characterized flow cytometer was used to determine the inherent fluorescent CV of dimly fluorescing beads. The fluorescence from the beads is also calibrated in terms of molecules of equivalent soluble fluorophore (MESF). The beads with known inherent CV and MESF provide a standard against which the instrument contribution to the CV of dim fluorescence can be measured. By measuring the standard deviation (SD) of the fluorescence histogram from unstained beads (noise) we obtain a second measure of instrument performance. The bead CV and noise SD are a sufficient pair of factors to determine the optical capability of a flow cytometer to resolve dim subpopulations of particles. It is also possible to use the measurements to calculate B and Q and use this information to predict the shapes of fluorescence histogram distributions of dim particles.