Evaluating fluorescence sensitivity on flow cytometers: an overview

The current paradigms for assessing fluorescence sensitivity on flow cytometers do not provide an adequate assessment of an instrument's ability to detect and measure weak fluorescence on stained particles. The capability to resolve dimly stained populations depends on two factors: the background noise (B), and the efficiency (Q) with which the fluorescence from the fluorochrome molecules are converted to photoelectrons. Any single statistical measure of fluorescence histogram distributions will be unable to uniquely characterize an instrument. Therefore, neither of the routinely used methods (detection threshold and delta channel) measure sensitivity completely and unambiguously. We show the limitations of these methods and propose that instrument sensitivity be characterized in terms of both background noise and detection efficiency in order to determine better the capability to detect and resolve weakly fluorescent particles.