Is there a Pot of Gold at the End of the Spectrum?

FOR many years, the convention for flow cytometry has been the measurement of one fluorochrome per detector. As the need for more markers to define unique subpopulations has grown, flow cytometers have addressed this need with more lasers and more detectors to increase the number of fluorochromes that can be used in a single panel. The development of new fluorochromes to fill up these detectors off additional laser lines soon followed (1,2), leading to the expansion of panels from 8–12 colors (3,4) to 17–28 colors (5,6). However, the challenge of maintaining population resolutions as these panels grew larger was a new reality due to the issues of spillover spreading (SS) associated with fluorescence (7), as well as a limit to the number of fluorochromes that could be run in the same panel together. Niewold et al. present in this issue (page 1165–1179) the possibilities of another approach using full spectrum flow cytometry, which utilizes many detectors and multiple excitation sources to define each fluorochrome, a paradigm shift from the one fluorochrome one detector legacy (Fig. 1A). The idea of spectral cytometry is not new. The first reports of measuring fluorescence spectra in a flow cytometer were published as early as the late 1970s (8) and then again in the mid-1990s (9). In 2004, Robinson et al. published the use of a multi-photomultiplier (PMT) array for the measurement of spectrally dispersed light, which was later patented and licensed by SONY, Inc. (SONY Biotechnology, San Jose, CA) (10). This led to the first commercial spectral flow cytometer by SONY (SP6800) in 2013. Over the years, there have been multiple approaches which have taken advantage of using more than a single detector to define the emission spectrum of a fluorochrome (11-13). Recently, with the release of the Cytek Aurora (Cytek Biosciences, Inc., Fremont, CA) in 2017, a commercial flow cytometer was implemented which took advantage of the full spectrum of fluorescence emission across all excitation sources, while also leveraging the increased quantum efficiency of avalanche photodiodes, especially in the range of red to far-red emission wavelengths (14). There are real advantages to cross laser excitation and the collection of these emissions. In some cases, where fluorochromes are close to identical in the area of their peak emission, there may be differences in their emission from the non-peak excitation sources (Fig. 1B). This information can be used to distinguish two fluorochromes that have very similar peak emissions, thus enabling an unmixing algorithm to classify the two fluorochromes as unique. As a result, the ability to provide a full spectrum signature, across multiple lasers and a large array of detectors, is expanding the possibilities of panel design to break the fluorescence ceiling of 28 colors. It should also be noted that, in addition to the number of detectors, the quality of the light detection also plays a role in reducing SS and improving resolution. The use of better photodetection approaches with increased quantum efficiency across the spectrum and methods to reduce noise will serve to produce higher quality raw data (increased signal:noise) to use in unmixing algorithms (13). In this issue (page 1165–1179), Niewold et al. present a comparison between two conventional flow cytometers (Fortessa X-20 and LSR-II) and a full spectrum flow cytometer (Aurora). The authors highlight some very key advantages of the full spectrum approach; the ability to resolve and employ highly overlapping dyes in a panel to expand the number of usable fluorochromes within a panel, the reduction in SS and hence increase in population resolution, and the utility of defining the spectrum of the autofluorescence of cells within an experiment. The authors nicely demonstrated that the increase in population resolution and decreased SS was more than just the result of the method that was used to either compensate or unmixed the multicolor samples. Here, they compared data from a conventional system using compensation and data from the full spectrum system using either the ordinary least squares spectral unmixing algorithm provided by the instrument manufacturer or compensation of the full spectrum data using peak detector or Flow Cytometry Support Services, LLC, Alexandria, Virginia, 22314, USA