Flow cytometry has seen the light: All of it.

Abstract

For much of its history, flow cytometry has selectively collected emitted bands of wavelengths of light, while giving up much of the valuable information outside of those bands. This approach, while practical and effective for panels of just a few fluorophores, limits the information available and the ability to more clearly define the spectral fingerprint associated with any given fluorophore. To expand the number of fluorophores that can be multiplexed in a panel or provide the ability to measure small spectral shifts in fluorophores associated with functional probes, a more detailed collection of spectral emission across an array of lasers is required. Furthermore, with this more detailed spectral information, the ability to characterize the autofluorescence profiles of a variety of unlabeled cell types has opened additional opportunities. Recent advances in flow cytometers have enabled the collection of very narrow bands of light across the entire spectrum resulting from the excitation of multiple laser wavelengths. By doing so, the identification of a “spectral fingerprint” that is unique to each unique fluorophore becomes a way to better differentiate one fluorophore from another, even if they only differ slightly [1]. This new approach has become to be known as full-spectrum flow cytometry (FSFC) and this Special Issue is dedicated to highlighting some of the advancing applications that this technology has recently enabled. Spectral Cytometry itself is not new. As detailed in the historical perspective of measuring the full spectrum to define a source of emitted light, Nolan (DOI: 10.1002/cyto.a.24566—Part One pp.812–817) points out the rich history of the developments of pioneers that have led to recent advances in instrumentation, optics, and reagent development. In this review, the author highlights the origins and early milestones, and provides a description of current instrumentation as well as the potential for the future of the technology and associated applications. As with conventional approaches, mathematical approaches are used to define and quantitate a spectral emission so the proportional representation of each fluorophore in a multiplexed environment can be determined. In conventional flow cytometry (CFC), this is known as fluorescence compensation; in FSFC this process is referred to as spectral unmixing. While both these processes accomplish similar goals, there are some specific nuances associated with each approach. In this Special Issue, Novo (DOI: 10.1002/cyto.a.24669—Part Two next issue) summarizes some of these differences to help the reader understand the similarities and differences between these two approaches and highlights the value of having additional measurements from which to calculate abundances. As with many other technological advances, new opportunities have arisen to advance the applications of flow cytometry with the advantages of FSFC. Researchers are beginning to develop optimized multicolor immunophenotyping panels (OMIP) using FSFC to expand the depth of understanding and characterization of the cellular immune system. The first OMIP utilizing FSFC was published in 2020, which presented a 40-marker panel to comprehensively phenotype major cell subsets in blood [2, 3] published OMIP 83, which utilized FSFC to delineate peripheral blood monocyte subsets as well as identify phenotypic markers across a variety of functional states. Also in this Special Issue, Barros-Martins et al. (DOI: 10.1002/cyto.a.24564—Part One pp.856–861 present OMIP 84: 28-color FSFC panel for the comprehensive analysis of human γδ T cells. In this OMIP, the authors provide an optimized panel for the phenotyping of all main human γδ T-cell subsets utilizing V genes of the T-cell receptor and provide a list of phenotypic surface receptor markers to further characterize the stages of cellular maturation. While panels utilizing FSFC for the in-depth profiling of immune subsets continue to be developed, there is also interest in already established immune monitoring panels developed for other high-dimensional single-cell technologies such as mass cytometry. Jaimes et al. (DOI: 10.1002/cyto.a.24565—Part Two next issue) compare FSFC and mass cytometry, using an established 32-marker mass cytometry panel to demonstrate the two technologies can obtain comparable results. This comparison is valuable for those who wish to utilize existing panels originally developed for studies using mass cytometry but may not have access to this technology. One of the common approaches for achieving high throughput of multiplexed panels, while reducing the sample-to-sample variability due to staining and acquisition of different samples over time is the use of cellular barcoding. [3-8] Juncker et al. (DOI: 10.1002/cyto.a.24543—Part Two next issue) address this issue in FSFC with fluorescence bar coding using anti-CD45, allowing for the simultaneous labeling and acquisition of up to 20 different live PBMC samples. The authors report efficient and robust de-barcoding using a standard Boolean gating approach. With the use of different fluorescently conjugated anti-CD45 antibodies as the mechanism for barcoding, the authors were able to demonstrate analysis of complex live PBMC populations without introducing major batch effects. Another important highlight in this Special Issue is the publication of several new Phenotype Reports, which took advantage of FSFC technology. This manuscript format is meant to be concise and provide a list of widely accepted and well-characterized markers, antibody clones, and gating strategies to identify specific cell populations. Shenoy et al. (DOI: 10.1002/cyto.a.24522—Part Two next issue) leveraged the power of FSFC and the use of high-dimensional clustering algorithms to identify a new level of heterogeneity in the adaptive immune environment of resident murine lung, including unexpected subsets of T and B resident memory cells. Likewise, Vanuytsel et al. (DOI: 10.1002/cyto.a.24540—Part Two next issue) utilized FSFC to incorporate the increasing number of unique markers expressed throughout the developmental process of hematopoietic stem cells (HSC) and multipotent progenitor cells (MPP) to comprehensively profile the HSC compartment in the human fetal liver. Other novel applications are also benefitting from the unique advantages of FSFC. For example, Henderson et al. (DOI: 10.1002/cyto.a.24472—Part One pp.818–834) have developed an improved method of resolution for detecting Förster resonance energy transfer (FRET) in living cells. Utilizing both CFC and FSFC, these authors were able to improve the resolution of the FRET detection while also adding additional markers to phenotype the specific cells being measured, without any loss of resolution. By doing so, they were able to measure biological processes in real time, opening the opportunity for the potential to sort cells based on FRET. As mentioned previously, the capacity to produce unique spectral fingerprints of the autofluorescence of different cell types provides a very powerful tool. In some cases, identifying the autofluorescence signatures and separating them from the spectral signatures of fluorophore-labeled markers (autofluorescence extraction) enhances resolution and specific population resolution. In this issue, Jameson et al. (DOI: 10.1002/cyto.a.24555—Part Two next issue) take an in-depth look at the identification of autofluorescence and its impact on the detection of dim fluorescent signals and the identification of specific cell subsets with low-abundance antigen expression. Using mathematical model predictions and high dimensionality reduction algorithms, these authors were able to demonstrate cellular distribution of a weakly expressed fluorescent protein that reports on a low-abundance immunological gene. Likewise, Kharraz et al. (DOI: 10.1002/cyto.a.24568—Part One pp.862–876) enhance the identification of myeloid resident cells in regenerating muscles cells by utilizing these autofluorescent signatures as additional fluorochromes in the unmixing algorithm. With this method, the authors were able to overcome the typical technological limitations in flow cytometry due to highly autofluorescent cell populations. Finally, Peixoto et al. (DOI: 10.1002/cyto.a.24567—Part Two of next issue) highlight the use of an automated autofluorescence finder tool to uniquely identify autofluorescent populations of stromal cells in the fetal liver. Using this approach, they were able to identify two unique signatures that were attributable to three distinct cell types and subsequently identified surface markers that characterize these populations. It is clear from these multiple publications that autofluorescence characterization using FSFC is a very powerful discovery tool that can enhance the identification and characterization of otherwise difficult cell and tissue sample types by flow cytometry. While FSFC has been predominately used to enhance experimental research, there is also a great opportunity within the clinical diagnostic arena to exploit the ability to expand the numbers of fluorophores that can be used in a single tube for clinical diagnosis and prognosis of blood-related disorders. There are several reasons this is desirable: (1) sample size can often be limited; (2) more in-depth initial immunophenotyping can aid in the monitoring of the progression of disease, such as in minimal residual disease (MRD); and (3) full characterization in a single tube eliminates the need for marker redundancy across tubes and therefore is more cost-effective [9]. In this Special Issue, Soh et al. (DOI: 10.1002/cyto.a.24667—Part Two next issue) describe a 27-color FSFC panel for the detection of MRD in acute myeloid leukemia (AML). In this publication, the authors investigate the feasibility of using an expanded panel, which incorporates all the existing recommended markers and compared the panel to the commonly used 8-color panel. Their findings of a high concordance with the existing method and the ability to achieve the required minimum detection sensitivities of 0.1% provide a promising alternative approach for the diagnosis of MRD. While many new applications that take advantage of the power of FSFC are presented in the issue, likewise new tools are being developed to enable researchers to continue to develop these applications. For example, the development of new and novel fluorochromes continues to be an important developmental area for FSFC. Seong et al. (DOI: 10.1002/cyto.a.24537—Part One pp. 835–845) address the limited availability of fluorophores emitting in the >800 nm range. The authors present the development of six new and novel infrared dyes using PE and APC tandems, using a typical FRET approach by covalently linking a protein donor dye with an organic small molecule acceptor dye. These new dyes were compatible with fixation/permeabilization protocols and were relatively bright and stable when conjugated with monoclonal antibodies. The addition of such new fluorophores provides opportunities for expanded panels for full-spectrum flow cytometers with fewer lasers. Another very important application of FSFC is the detection of multiple fluorescent proteins (FPs), especially when their peak emission is very similar, such as with GFP and YFP and their derivatives [10]. However, single-color controls for many of these FPs are not readily available, and often require maintaining cell lines stably expressing each FP, along with the non-expressing parental cell line. Furthermore, in animal models with tissue-specific FP expression, the need for wild-type animals is also required as a control. Monard (DOI: 10.1002/cyto.a.24557—Part One pp.846–855) addresses these limitations by producing purified FPs, which are then coupled to polystyrene microspheres. These FP microspheres are ready to use and stable at 4°C without fluorescence degradation. The manuscript provides detailed protocols, which can be duplicated by other labs without any special skills or equipment. I think it is abundantly clear that the future of FSFC is bright (and colorful). The number of publications continues to increase, with well over 900 publications to date. The value of the FS approach has been documented in several publications [1, 11, 14] and likewise so has the development of detailed protocols. [15, 16] Researchers are only beginning to scratch the surface of what is possible with FSFC, and as more unique and functional fluorescent probes become available, the greater impact this technology will have on scientific discovery. The compilation of manuscripts in this Special Issue is just the beginning of what is to come, leading to what appears to be a bright and colorful time in flow cytometry.