Response to: Comments on the publication “Determination of Background, Signal‐to‐Noise, and Dynamic Range of a Flow Cytometer: A Novel Practical Method for Instrument Characterization and Standardization” by Giesecke et al. Article Swipe
YOU?
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· 2020
· Open Access
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· DOI: https://doi.org/10.1002/cyto.a.23977
· OA: W3003223785
In the September 2017 issue of Cytometry A, we have described the use of an LED pulser to characterize a flow cytometer's photodetector's response curve over a defined range of amplification, henceforth referred to as the signal-to-background ratio (SBR) method 1. This approach aims at characterizing the amplification characteristics of the photodetectors of the flow cytometer, based on defined LED pulses. We have described how to deduct signal-to-background (noise) ratios (SBR) and dynamic range (DNR) curves from these data. The SBR method enables the user to determine the optimal operating point of a photodetector without the use of beads. By combining the SBR method with beads, the excitation characteristics of the laser, and the detection characteristics of the photomultipliers can be determined and optimized independently of each other. We highly appreciate the commentary of James Wood, concerning the appropriateness of using either signal height or signal area in determining the amplification characteristics of photomultipliers. We are in complete agreement that the area signal can be used to this end, in particular for the measurement of cells. The point we want to highlight is that in the LED-based SBR approach (which is only concerned with the characterization of PMT response), the use of signal height is entirely justified. Moreover, it offers a number of advantages, as discussed in the original publication 1: The laser-light induced background (LIB) signal can be distinguished from the noise (PMT dark current) better by the measurement of the height than the area of the signal, as discussed in the original paper. The amplitude of the signal of the LIB is not influenced by the noise 2. The area of the noise, however, influences the area of the LIB, because they are overlapping 1. Furthermore, the calibration factor K, which we use for the scale calibration is obtained under conditions where the measured variation (CVmeasured), is significantly higher than the variations of LIB, noise, and LED (CVbackground(LIB), CVelectronic noise, CVLED intrinsic) as we have outlined in detail in the publication (page 7 1). As we show in the publication, the experimental data (fig. 3 1) then confirm these theoretical considerations. In summary, the SBR method expands the established toolbox for instrument setup and is not intended to override previous work. In combination with the bead-based methods, it enables the evaluation of photomultiplier properties independent of the laser excitation properties and thus the quantitative comparison of flow cytometers.
