Fluorescent Probe Technologies Suitable for Multicolor Spectral Flow Cytometry

Abstract

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Flow cytometry is a versatile tool that has applications in numerous biomedical research disciplines, including cancer biology, immunology, infectious disease monitoring, microbiology, and molecular biology. Instrumentation for performing flow cytometry has advanced considerably over the past several decades. Systems incorporating multiple lasers are commonplace, as are specialized instruments designed for particular applications, such as systems using 96-well sample loaders, systems combining microscopy with flow cytometry, and systems combining mass spectrometry with flow cytometry. Spectral flow cytometry measures the entire fluorescence emission spectra for each fluorophore deployed in a multicolor labeling experiment to produce unique spectral fingerprints. Subsequently, each spectrum is unmixed to provide a pure signal for each fluorophore. Such complete spectral analysis is beginning to supplant conventional photomultiplier tube (PMT)-based approaches as the preferred detection approach for high-dimensional multicolor flow cytometry. This technology shift provides an excellent opportunity to implement comprehensive, high-performance fluorophore families spanning the UV-visible-NIR light spectrum.

Multicolor Spectral Flow Cytometry Instrumentation

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Flow cytometry is an analytical method that provides rapid, multi-parametric analysis of thousands to millions of individual cells in solution (McKinnon, 2018). Particular phenotypic characteristics of individual cells within a cell population are analyzed based upon their fluorescent and light scattering characteristics. Flow cytometry is commonly used in biomedical research and the analysis of clinical specimens. Flow cytometers employ lasers as light sources to generate both scattered and fluorescent signals that are registered by detectors, such as photodiodes or photomultiplier tubes. These analog signals are then converted into electronic signals, analyzed by a computer, and written to a standardized data file format (.fcs).

In order to expand upon the number of fluorophores employed in a flow cytometry experiment beyond ~28, a pretty high level of detail is required to distinguish among fluorophores whose spectral emission signatures are similar. The required level of detail demands high-quality signals, low noise, excitation-specific full emission profiles, and vigilant panel design and optimization. Spectral flow cytometry represents a novel flow cytometry technology platform offering significantly improved multiplexing capabilities relative to traditional flow cytometry (Robinson, 2019; Fox et al., 2020). While in some instances, conventional flow cytometers are capable of detecting panels containing as many as two dozen or so different fluorophores, spectral flow cytometers can distinguish as many as 40 different fluorophores using a single multi-parametric panel.

Spectral flow cytometry shares much of the same hardware that is associated with conventional flow cytometry. Both systems employ standard fluidic and laser technologies that enable cell-by-cell spectral analysis. For both spectral and conventional flow cytometry methods, the process begins by delivering a sample stream through a flow chamber where cells move in single file at a constant velocity, a process referred to as hydrodynamic focusing. This allows uniform and efficient excitation by a set of monochromatic lasers. Emitted photons are then detected and analyzed by a combination of optics and software to identify unique spectral emission information from fluorophore-labeled biomolecules.

Spectral flow cytometry differs noticeably from traditional flow cytometry when considering its optical configuration and the data analysis software (figure 1). With conventional flow cytometry, photons emitted by a particular fluorophore are directed through a series of dichroic mirrors and bandpass filters to partition the light into a narrow bandwidth for detection by a specific photomultiplier tube (PMT). However, spectral flow cytometry is different in that it employs dispersive optics, such as prisms or spectrographs to disperse photons based upon wavelength across an array of detectors. This approach broadens a fluorophore's spectral profile by capturing the entire visible and near-IR spectrum of light, allowing for higher resolution spectral analysis relative to the aforementioned traditional optical configurations, which detect only a tiny portion of the emission spectra. As a result, fluorophores with similar emission spectral profiles that are challenging to differentiate by traditional flow cytometry can readily be distinguished using spectral flow cytometry. As many as 40 different fluorophores can be analyzed, including those with emission spectra in close proximity to one other.



Despite its superior ability to distinguish between fluorophores, it is advisable to use fundamental flow cytometry principles in panel construction and avoid spectral spillover where possible when designing spectral flow cytometry experiments. Designing robust multicolor fluorescence panels entails vigilant consideration of fluorophore spectral properties, choosing those spectral profiles that are best separated by the instrument. Using traditional flow cytometry, any multi-parametric panel containing a dozen or more fluorophores will undoubtedly have some overlap of emission spectra, resulting in noise in channels from emissions of unintended fluorophores (i.e., spectral spillover). To account for this, single-color control samples must be included with each experiment to ascertain the level of spectral overlap encountered in each detector. Then, with traditional flow cytometry, a mathematical approach referred to as compensation is applied using the controls to subtract overlapping spectra and effectively isolate a fluorophore's emission profile. This is achieved with a correction factor obtained using the ratio of the intended and unintended fluorophores emitting into a particular channel.

Mathematical models are required with spectral flow cytometry to correct for any spectral spillover among channels. This is necessary since the instrument must distinguish between multiple fluorescent profiles across the entire visible light spectrum rather than from a few distinct channels. The process of deconvoluting fluorophore emission spectra across an array of detectors is known as spectral unmixing. This form of compensation also requires experimental reference controls as well as noise-reducing mathematical algorithms, such as the least-squares method. This approach is especially valuable when interrogating cell culture samples, which are susceptible to a high degree of autofluorescence.

The Cytek Aurora benchtop flow cytometer (Fremont, CA) is a high-performance spectral flow cytometer that the AAT Bioquest team has extensive experience using in conjunction with our comprehensive suite of fluorophores and fluorescent proteins. The instrument leverages full-spectrum technology to enable the use of a wide range of novel fluorophore combinations without reconfiguring the system for each application. The instrument generates high-resolution data at the single-cell level, facilitating the resolution of even the most challenging cell populations, including cells exhibiting high autofluorescence or cells expressing low levels of target biomarkers, even in the context of complex multi-analyte detection. Their SpectroFlo^® software provides an intuitive workflow that guides users from QC to data analysis with tools that simplify performing even the most challenging applications.

With up to five lasers, three scattering channels, and 64 fluorescence channels, the Aurora system is highly flexible, intuitive, and ultra-sensitive. With its intuitive optical design, compact footprint, and upgradeability from 3 to 5 laser configurations, the Aurora system suits every laboratory's needs, from simple to high complexity applications.

The system's state-of-the-art optics and low-noise electronics provide excellent sensitivity and resolution. Flat-top beam profiles, combined with a uniquely designed fluidics system, translate to outstanding instrument performance, even at high sample flow rates.

Optimized Multicolor Immunofluorescence Panels for Multicolor Spectral Flow Cytometry

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A wide range of fluorescent reagents is employed in flow cytometry, including fluorescent dye-conjugated antibodies, DNA binding dyes, viability dyes, ion indicator dyes, and different fluorescent proteins. By collecting substantially more information than conventional flow cytometry concerning each cell in a sample, full-spectrum multicolor flow cytometry has become the platform of choice for developing optimized multicolor immunofluorescence panels (OMIPs). Numerous OMIPs have been published in the literature over the years, obviating the need for extensive time to independently design such panels (Mahnke et al., 2010; Park et al., 2020). These published OMIPs also serve as valuable starting points for creating novel OMIPs and provide a mechanism for recognizing panel developers through citation of their work.


As an impressive example of a biomedical research problem requiring the application of OMIPs, elucidating the intricate details of the human immune response requires the capacity to perform high-throughput, in-depth analysis at both the single-cell and population levels. OMIP-069 is a recently developed forty-color full spectrum flow cytometry panel designed for the deep immunophenotyping of major cell subsets in human peripheral blood, including subsets of T, B, NK, NKT, monocyte, and dendritic cells (Park et al., 2020). The cited panel shares similarities with OMIPs -015, -023, -024, -030, -033, -034, -042, -50, -058, -063, which were designed to identify the major leukocyte subsets in human blood, but also partially overlaps with OMIPs -013, -017, -021, -030, and -060 to facilitate characterization of T cells; OMIPs -004, -006, and -015 for Treg immunophenotyping; OMIP -044 for dendritic cells; OMIPs-003, -033, and -051 for B cells; and OMIPs -029, and -039 for NK cells. The panel includes a live-dead viability stain, a compendium of organic fluorophores, and various fluorescent proteins, such as allophycocyanin (APC), peridinin-chlorophyll-protein (PerCP), and various phycoerythrin (PE)-dye conjugates. The cited 40-color panel provides a potent approach for the in-depth characterization of lymphocytes, monocytes, and dendritic cells in human peripheral blood. The panel is suitable for interrogating nearly the entire cellular composition of the human peripheral immune system and should be particularly valuable for studies wherein sample availability is limited or unique biomarker signatures are being pursued.

Selecting Fluorophores for the Development of Multicolor Phenotypic Panels

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In flow cytometry methods development, it is critically important to be familiar with the configuration of the instrument that a particular phenotypic panel is being designed for. The number and types of lasers and filters that the instrument is equipped with dictate which fluorophores can be deployed in the panel. Fluorophores are selected that have excitation maxima closely matching the lasers in the system, while for conventional flow cytometry filters should be designed to detect the targeted fluorophore's emission wavelength maximum without registering light from other laser sources in the instrument. For multicolor spectral flow cytometry, an entire emission profile is detected, and fluorophores should be readily distinguishable on this basis. Typically, when a panel of fluorophores is required, distributing fluorophores as widely as possible across the excitation and emission spectra are recommended to minimize interference.

To minimize any signal spillover, which can adversely influence resolution and sensitivity, bright fluorophores, such as PE and APC, should be used with low abundance phenotypic targets, and organic fluorophores, such as mFluor™ dyes should be paired with more highly expressed targets. This improves the ability of the flow cytometer to discriminate between specific signal and background fluorescence arising from variables such as non-specific staining and cellular autofluorescence. In cases wherein an antibody is not commercially available bound to the desired fluorophore, ATT Bioquest offers custom antibody labeling kits and services.

The mFluor™ Dyes

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As briefly summarized above, the foundation for developing multicolor phenotypic panels, such as the OMIPs, is a high-performance series of reactive organic fluorophores, fluorescent proteins, and tandem fluorophore-fluorescent protein conjugates that span the UV to near-infrared (NIR) region of the light spectrum. Designing and optimizing these panels can be a time-consuming and challenging activity. Several hurdles must be overcome, such as qualification (e.g., titration) of individual reagents, evaluating any potential interactions between reagents selected for a particular panel, choosing the best combination of fluorophore-antibody pairings, and overcoming the sensitivity losses inherent in multicolor fluorescence experiments. The design and optimization phase of creating even a modest 12 color panel may require 2–4 months of effort before the panel is mature enough for actual experimental applications. As a dedicated vendor in this specialized area of reagents, AAT Bioquest offers in-house developed conjugates, including custom-synthesized conjugates, to assist investigators in achieving the high-level multiplexing required for the development of multicolor immunofluorescence panels.

Developed exclusively by AAT Bioquest, mFluor™ dyes exhibit excellent aqueous solubility and large Stokes Shifts, and their hydrophilic nature minimizes the need for employing organic solvents (Table 3). Currently, we offer 26 unique mFluor™ SE dyes. mFluor™ dyes use the following naming convention: 'mFluor™ + Excitation Laser + Emission Wavelength + Reactivity'. For example, mFluor™ UV460 SE (Ex/Em max = 364/461) is a UV light-excitable succinimidyl ester dye that emits around 460 nm.

mFluor™ dyes have been used extensively to label antibodies proteins and other biomolecules for multicolor flow cytometry applications. The absorbance maxima of mFluor™ dyes are designed to be optimally excited by one of the major laser lines commonly equipped in flow cytometers, such as the 350 nm, 405 nm, 488 nm, 532 nm, 561-568 nm, or 633-647 nm laser lines. In conjunction with phycobiliproteins PE, APC, and their tandems, mFluor™ dyes are excellent fluorophores for immunophenotyping (figure 2), FACS, and other flow cytometry-based applications. mFluor™ dyes are available in a wide selection of products, including reactive dyes and antibody labeling kits, as well as mFluor™ streptavidin conjugates for signal amplification and annexin V-mFluor™ conjugates for apoptosis detection.



One crucial advantage of mFluor™ reactive dyes is that they can be covalently labeled to biomolecules without self-quenching, which results in intensely bright fluorescent conjugates. mFluor™ reactive dye formats include amine-reactive succinimidyl esters (SE) and thiol-reactive maleimides for labeling antibodies and proteins. Additionally, mFluor™ dyes are available as their acid form for labeling proteins, peptides, amine-modified oligonucleotides, and other amine-containing biomolecules using carbodiimide conjugation chemistry (EDAC).

To ensure stable, dependable antibody labeling for a variety of flow cytometry applications, antibody labeling kits can be an optimum choice for preparing the required antibody conjugates. ReadiLink™ Rapid mFluor™ Antibody Labeling Kits from AAT Bioquest provide a convenient method for labeling microscale volumes of antibodies with our superior mFluor™ dyes. The unique chemistry of ReadiLink™ kits enables researchers to effortlessly label and recover 100% of their antibodies without a purification step. Since ReadiLink™ mFluor™ conjugates are covalently labeled, they are stable for long-term storage and are ideal for demanding applications including immunophenotyping, multiplex flow cytometry, FACS, and other flow-based applications. Also available are ReadiLink™ Rapid and xtra Rapid Antibody Labeling kits for conjugation of mFluor™ dyes, Alexa Fluor^® dyes, other fluorescent dyes, biotin, BSA, and KLH.

mFluor™ dyes provide simple, quick, and robust labeling of biomolecules, with high conjugation yields. The fluorophores offer superior photostability, solubility, and brightness generating strong fluorescence emissions over a broad pH range with little pH sensitivity and low spillover into most PMT detectors. As such, mFluor™ dyes offer excellent choices and flexibility for the design of multicolor phenotypic panels.

1. Extinction coefficient (M^-1cm^-1)
2. Quantum yield measured in aqueous solution

Phycobiliproteins

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Phycobiliproteins are photosynthetic light-harvesting proteins obtained from microalgae and cyanobacteria. This family of proteins contains covalently attached linear tetrapyrrole groups, referred to as phycobilins, which are involved in capturing light energy. In nature, energy absorbed by these phycobilins is efficiently transferred, by the process of fluorescence resonance energy transfer (FRET), to chlorophyll pigments for use in the photosynthetic process. Since phycobiliproteins possess extremely high fluorescence quantum yields and absorbance coefficients (e.g., molar extinction coefficients) over a wide range of the light spectrum, they are exceedingly fluorescent and thus are considered an excellent reagent for use in fluorescence applications, especially in flow cytometry (Table 4).

Phycoerythrin (PE), allophycocyanin (APC), and their tandem fluorophore conjugates are among the most suitable phycobiliproteins for flow cytometry applications. Conjugation of these dyes to macromolecules with biological specificities, such as antibodies, protein A or streptavidin, can be used in fluorescence-based detection applications that require high sensitivity but not necessarily photostability, such as fluorescence-activated cell sorting (FACS) and immunophenotyping. Compared with organic and synthetic fluorescent dyes, the advantages of phycobiliproteins as fluorescent labels include; long-wavelength fluorescence excitation and emission to minimize interference by auto-fluorescence from biological materials, minimal fluorescence quenching, high water-solubility, significant Stokes shifts with well-resolved emission spectra for multicolor analysis and multiple sites providing for stable conjugation with organic and synthetic compounds, including antibodies, cyanine dyes, iFluor^® dyes, and mFluor™ dyes.

Tandem Dyes Further Expand Options for Multicolor Immunofluorescence Panels

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When developing high-plex phenotypic panels, the use of spectrally similar fluorophores becomes practically unavoidable. One solution to this problem is to separate the complex fluorophore combinations by segregating them into cellular subpopulations that are gated and analyzed separately, thus limiting spectral spillover between populations. Another helpful approach for increasing panel size and diversity is by employing tandem dyes.

Tandem dyes comprise of two different fluorophores, a fluorescent donor and a longer-wavelength emitting fluorescence acceptor, that are conjugated to the same biomolecule. One fluorophore in the pair transmits energy to the other by the process of fluorescence resonance energy transfer (FRET). Since the second fluorophore emits light at a higher wavelength than that emitted by the first fluorophore, the number of fluorophores that can be distinguished using the same laser for excitation is effectively increased. Typically, PE and APC are used as donor fluorophores when generating tandem dyes, as illustrated with APC-iFluor^® 700 and PE-iFluor^® 750. In flow cytometry, tandem dyes are particularly suited for multicolor analysis of cells due to their exploitation of a single excitation source and their significantly large Stokes shifts.

PerCP (Peridinin-chlorophyll-protein complex), obtained from Dinophyceae spinosum, has an extremely high extinction coefficient, a high quantum efficiency, and a large Stokes shift. It is especially well excited by the 488 nm argon-ion laser, with its maximum emission peak at 677 nm. PerCP protein is commonly employed for fluorescent immunolabeling, especially fluorescent-activated cell sorting (FACS). Tandem conjugates with cyanine dyes, such as PerCP-Cy5.5, can be excited with a standard 488 nm laser and emits in the far red at a longer wavelength for multicolor flow cytometric analysis of cells. These multiple emission wavelengths make PerCP-cyanine conjugates potentially useful fluorochromes for multicolor analysis with FITC, PE, and other fluorophores. AAT Bioquest offers iFluor^® and mFluor™ protein labeling dyes, which are generally brighter and more photostable than the corresponding cyanine dyes of similar wavelengths.

Table. 5 24-Color panel for identifying circulating cell subsets in human peripheral blood

Specificity	Violet Laser Fluorochromes	Specificity	Blue Laser Fluorochromes	Specificity	Red Laser Fluorochromes
CCR7	mFluor™ Violet 420	CD11c	XFD488 (Alexa Fluor® 488 Equivalent)	CD27	APC
CD19	mFluor™ UV420	CD45RA	iFluor® 488	CD123	iFluor® 647
CD16	mFluor™ Violet 450	CD3	iFluor® 532	CD127	APC-iFluor® 700
TCR gamma delta	mFluor™ Violet 500	CD25	PE	HLA DR	APC-iFluor® 750
CD14	mFluor™ Violet 510	IgD	PE-iFluor® 594
CD8	mFluor™ Violet 590	CD95	PE-Cy5
CD1c	mFluor™ Violet 610	CD11b	PerCP-Cy5.5
PD-1	Brilliant Violet 650™	CD38	PerCP-eFluor® 710
CD56	Brilliant Violet 711™	CD57	PE-Cy7
CD4	Brilliant Violet 750™
CD28	Brilliant Violet 785™

1. Brilliant Violet™ is a trademark of Sirigen Group Ltd.
2. eFluor^® is a trademark of Thermo Fisher Scientific

Conclusion

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The requirement to simultaneously interrogate increasingly more phenotypic features in complex cell populations, such as human peripheral blood cells, has demanded the implementation of more powerful instrumentation adaptations beyond those found in traditional flow cytometry. One important advance in multicolor immunofluorescence analysis has been the introduction of full spectrum multicolor spectral flow cytometry. This technology captures the entire emission spectrum of fluorophores using arrays of highly sensitive light detectors and has enabled simultaneous characterization of as many as 40 parameters in a single sample (Park et al., 2020). Implementation and broad adoption of this new flow cytometry-based instrumentation require careful design and validation of optimized multicolor immunofluorescence panels (OMIPs). These, in turn, require the availability of high-performance series of reactive organic fluorophores, fluorescent proteins, and tandem fluorophore-fluorescent protein conjugates that span the UV-visible-NIR region of the light spectrum.

References

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