A novel method for flow cytometer characterization by determination of detector background, signal-to-noise, and dynamic range
Claudia Giesecke1,2, Kristen Feher3, Konrad von Volkmann4, Jenny Kirsch3, Andreas Radbruch1 and Toralf Kaiser3
1Department of Cell Biology, Deutsches Rheuma-Forschungszentrum Berlin, ein Leibniz Institut, Berlin, Germany, 2Berlin-Brandenburg School for Regenerative Therapies (BSRT), Berlin; 3Department for Cytometry and Cellsorting, Deutsches Rheuma-Forschungszentrum Berlin, ein Leibniz Institut, Berlin, 4APE Angewandte Physik und Elektronik GmbH, Berlin, Germany
Accurate flow cytometer setup is fundamental to empower best experimental results. Regardless of the instrument used, maximum resolution of the populations of interest is the primary goal, meaning that ideally negative populations should be above noise or background, positive populations should be below the upper range limit and separation of the populations should be at maximum. In engineering sciences specific metrics are assigned to evaluate the lower detection limit, sensitivity and the upper detection limit at once, i.e. signal-to-noise ratio (SNR) and the dynamic range (DNR). Recent introduction of the quantiFlash®, a pulsed precision LED light source, now enables a new way of determination of these performance metrics for flow cytometry, independent of sample or bead preparation and instrumental factors not related to signal intensity. We used the quantiFlash® to characterize an instrument’s response to a stable input light signal over the entire PMT gain range. As a consequence, we propose a method to determine a flow cytometer’s SNR and DNR. This allows the selection of a voltage to optimize the signals delivered by the PMTs with respect to the background consisting of scattered laser light and electronic noise. Both contributions to the background vary depending on PMT voltage and are now ascertainable and distinguishable with the here proposed method. Such knowledge further allows for separation of technical and biological background which can help with experiment design. In conclusion, we introduce a new practical method for instrument sensitivity characterization, show how the optimal PMT voltage can be defined with respect to the SNR and DNR and discuss practical implications.