A well-defined scale calibration in flow cytometry can improve many aspects of data acquisition such as cytometer setup, instrument comparison and sample comparison. The theory for scale calibration was proposed by Steen over two decades ago, but it has never been put into regular use due to the lack of a widely available precision light source. The introduction of such a light source, the quantiFlash , gave this possibility. Here, we describe how this light source can be used to characterize a cytometer's PMT performance. We, therefore, characterized the instrument's response over the entire PMT voltage range. As a consequence, we propose a practical method to characterize a cytometer's signal-to-noise ratio (SNR) and dynamic range (DNR). This allows the selection of a voltage/gain corresponding to a PMT's maximum efficiency and hence the lowest electronic noise, which can help with experiment design. We further introduced a decibel (dB) scale for the presentation of SNR and DNR values. SNR and DNR are stand-alone values that allow the direct comparison of different instruments. Finally, with this method, it becomes clear that increased SNR comes at the expense of DNR and thus the limiting factor of modern cytometers is the DNR. © 2017 International Society for Advancement of Cytometry.

In recent years, multispectral flow cytometry systems have come to attention. They differ from conventional flow cytometers in two key ways: a multispectral flow cytometer collects the full spectral information at the single cell level and the detector configuration is fixed and not explicitly tuned to a particular staining panel. This brings about clear hardware advantages, as a closed system should be highly stable, and ease-of-use should be improved if used in conjunction with custom unmixing software. An open question remains: what are the benefits of multispectral over conventional flow cytometry in terms of sensitivity and resolution? To probe this, we use Q (detection efficiency) and B (background) values and develop a novel "multivariate population overlap factor" to characterize the cytometer performance. To verify the usefulness of our factor, we perform representative experiments and compare our overlap factor to Q and B. Finally, we conclude that the increased light collection of multispectral flow cytometry does indeed lead to increased sensitivity, an improved detection limit, and a higher resolution.

R scripts are available via https://fccf.mpiib-berlin.mpg.de/daten/drfz/bioinformatics/with{username,password}={bioinformatics,Sar=Gac4}.

Flow cytometers are robust and ubiquitous tools of biomedical research, as they enable high-throughput fluorescence-based multi-parametric analysis and sorting of single cells. However, analysis is often constrained by the availability of detection reagents or functional changes of cells caused by fluorescent staining. Here, we introduce MAPS-FC (multi-angle pulse shape flow cytometry), an approach that measures angle- and time-resolved scattered light for high-throughput cell characterization to circumvent the constraints of conventional flow cytometry. In order to derive cell-specific properties from the acquired pulse shapes, we developed a data analysis procedure based on wavelet transform and k-means clustering. We analyzed cell cycle stages of Jurkat and HEK293 cells by MAPS-FC and were able to assign cells to the G1, S, and G2/M phases without the need for fluorescent labeling. The results were validated by DNA staining and by sorting and re-analysis of isolated G1, S, and G2/M populations. Our results demonstrate that MAPS-FC can be used to determine cell properties that are otherwise only accessible by invasive labeling. This approach is technically compatible with conventional flow cytometers and paves the way for label-free cell sorting.

No abstract