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Spectral Flow Cytometry
Spectral flow cytometry represents a novel platform offering significant improvements in performance and multiplexing capabilities beyond conventional flow cytometry. By leveraging full-spectrum technology, spectral flow cytometry analysis awards researchers with unprecedented levels of flexibility, enabling the use of a wide range of novel fluorophore combinations (e.g., upwards of 40 different fluorophores) without reconfiguring the system for each application. The intuitive optical design and low-noise electronics of spectral flow cytometers, such as the Cytek Aurora or Sony Biotechnology SA3800 systems, deliver unparalleled sensitivity and resolution, regardless of assay complexity. Even the most challenging cell populations, including cells exhibiting high autofluorescence or cells expressing low levels of target biomarkers, can be resolved at the single-cell level. AAT Bioquest offers a comprehensive portfolio of assays and reagents to accommodate the needs of multiplexed spectral flow cytometry approaches in cell biology, cancer biology, and immunology. Choose from an assortment of high-performance iFluor® and mFluor™ dyes, PE and APC dyes, fluorescently labeled antibodies and other conjugates, DNA binding dyes, viability dyes, and ion indicators optimized for spectral flow analysis.
Introduction to Spectral Flow Cytometry
Spectral flow cytometry shares much of the same hardware that is associated with conventional flow cytometry. Like conventional cytometry systems, spectral cytometers utilize fluidic and laser technologies to enable cell-by-cell spectral analysis either by hydrodynamic focusing or through a microfluidics chip. In both systems, the process begins by delivering a sample stream through a flow chamber where cells move in a single file at a constant velocity. This allows uniform and efficient excitation by a set of monochromatic lasers. Data in the form of emitted photons are then detected and analyzed by a combination of optics and software to identify unique spectral emission information from the fluorophore-labeled biomolecules, as well as determine cellular properties related to size and granularity.
Comparison of conventional and spectral flow cytometer systems. A conventional flow cytometer relies on a series of bandpass filters and dichroic mirrors to separate light emissions into individual detectors. (Bottom) A spectral flow cytometer uses a grating or prism element to separate light into a focusing lens prior to detection. As separating light keeps diverging in space, a collimating lens is often used to parallelize and direct light linearly before reaching a detector.
Full Spectral Analysis
Where spectral flow cytometry most noticeably differs from conventional cytometry is in its optical design. Rather than using dichroic mirrors and bandpass filters to partition light emission into narrower bandwidths prior to detection, spectral flow cytometers use light dispersive optics, such as prisms or spectrographs, which disperse all emitted light across an array of detectors. This configuration 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 conventional design, which detects only a tiny portion of the emission spectra. As a result, fluorophores with similar emission spectral profiles that might have been challenging to differentiate by traditional flow cytometry, such as PerCP and PerCP-eFluor® 700, can be readily distinguished using spectral flow cytometry. The discrimination of these two red-emitting fluorophores is based on the differences in their overall emission spectra rather than detection in an individual channel (figure 2). As many as 40 different fluorophores can be analyzed, including those with emission spectra in close proximity to one other.
Fluorescence emission spectra of PerCP and PerCP-eFluor 710. Left: The emission spectra of PerCP and PerCP-eFluor 710, taken from Fluorescence Spectrum Viewer, demonstrates significant spectral overlap when using a single excitation and emission profile with conventional flow cytometry. Right: Full spectral analysis of PerCP and PerCP-eFluor 710 using a 5-laser spectral flow cytometer system. Although both share similar emission profiles, they exhibit unique full-spectrum signatures signals that allow the two fluorophores to be discriminated.
Compensation via Spectral Unmixing
Like conventional cytometry, spectral flow cytometry requires careful panel design and complex mathematical models 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 in spectral flow cytometry is referred to as spectral unmixing. This form of compensation requires single-stained reference controls, as well as noise-reducing mathematical algorithms, such as the least-squares method, to identify and separate unique spectral signatures within a complex mixture of emission signals in a multicolor experiment. This approach is especially valuable when interrogating cell culture samples, which are susceptible to a high degree of autofluorescence.
Table 1. Comparison between traditional and spectral flow cytometry.
| Parameter ▲ ▼ | Conventional Flow Cytometry ▲ ▼ | Spectral Flow Cytometry ▲ ▼ |
| Optical Hardware | Bandpass filters & dichroic mirrors Capture a narrow portion of a fluorophore’s emission spectrum | Dispersive optics capture entire fluorophore’s emission spectrum |
| Spectral Resolution | Acquires a narrow emission bandwidth from a single-laser excitation source | Acquires the entire fluorophore emission profile for each laser excitation source |
| Fluorophore Separation | Achieved by compensation (loss of some acquisition data) | Achieved by spectral unmixing (preserves more acquisition data) |
| Detection Sensitivity | Detection sensitivity is compromised by using compensation to address signal spill over | Superior detection sensitivity is assured using state-of-the-art optics and low noise electronics |
| Multiplexing Capability | Limited to perhaps 12 due to spectral overlap of fluorophores & limited number of detectors/filters installed in instrument | Superior spectral resolution so that as many as 40 fluorophore probes can be employed in a single analysis |
mFluor™ Dyes
Developed exclusively by AAT Bioquest, mFluor™ dyes exhibit excellent aqueous solubility, large Stokes Shifts, and their hydrophilic nature minimizes the need for employing organic solvents. Currently, 26 unique mFluor™ SE dyes are available for labeling antibodies and proteins. 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 and reacts with amine groups to form stable conjugates.
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, FACS, and other flow cytometry-based applications. mFluor™ dyes are available in a wide selection of products, including reactive dyes and ReadiLink™ antibody labeling kits, as well as mFluor™ streptavidin conjugates for signal amplification and annexin V-mFluor™ conjugates for apoptosis detection. 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.
One important advantage of mFluor™ reactive dyes is that they can be covalently labeled to biomolecules without self-quenching, which results in intensely bright fluorescent and photostable conjugates over a broad pH range. 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).
Table 2. mFluor™ active esters and kits for labeling antibodies, proteins and amine-modified biomolecules.
| mFluor™ Dye ▲ ▼ | Laser ▲ ▼ | Ex max ▲ ▼ | Em max ▲ ▼ | ε¹ ▲ ▼ | Φ² ▲ ▼ | CF @260 ▲ ▼ | CF @280 ▲ ▼ | Succinimidyl Ester ▲ ▼ | ReadiLink™ Kits ▲ ▼ |
| mFluor™ UV375 | UV | 351 | 387 | 30,000 | 0.94 | 0.099 | 0.138 | 1135 | |
| mFluor™ UV420 | UV | 353 | 421 | 80,000 | 1641 | ||||
| mFluor™ UV455 | UV | 357 | 461 | 20,000 | 0.42 | 0.651 | 0.406 | 1642 | |
| mFluor™ UV460 | UV | 358 | 456 | 15,000 | 0.86 | 0.35 | 0.134 | 1136 | |
| mFluor™ UV520 | UV | 370 | 524 | 80,000 | 0.03 | 0.495 | 0.518 | 1643 | |
| mFluor™ UV540 | UV | 373 | 560 | 90,000 | 0.35 | 0.634 | 0.463 | 1645 | |
| mFluor™ UV610 | UV | 371 | 609 | 90,000 | 0.25 | 0.949 | 0.904 | 1649 | |
| mFluor™ Violet 420 | Violet | 403 | 427 | 37,000 | 0.91 | 1105 | |||
| mFluor™ Violet 450 | Violet | 406 | 445 | 35,000 | 0.81 | 0.338 | 0.078 | 1150 | 1100 |
| mFluor™ Violet 500 | Violet | 410 | 501 | 25,000 | 0.81 | 0.769 | 0.365 | 1149 |
Phycobiliproteins
Phycobiliproteins - 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 to 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
Tandem dyes comprise two different fluorophores, a fluorescent donor and a longer-wavelength emitting fluorescence acceptor, that are conjugated to the same antibody. One fluorophore in the pair transmits energy to the other by 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) is another commonly used phycobiliprotein with an extremely high extinction coefficient, a high quantum efficiency, and a large Stokes shift. It is well excited by the argon-ion laser at 488 nm, with its maximum emission peak at 677nm. 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 3. PE, APC, and tandem dyes and kits for labeling antibodies, proteins, and other biomolecules.
| Phycobiliprotein ▲ ▼ | Laser ▲ ▼ | Ex max ▲ ▼ | Em max ▲ ▼ | Dye/Tandem¹ ▲ ▼ | ReadiUse™ Dye/Tandem² ▲ ▼ | Buccutite™ Antibody Labeling Kit ▲ ▼ |
| Phycoerythrin (PE) | Blue/Green/Yellow | 495, 546, 566 | 574 | 2558 (1 mg) 2556 (10 mg) 2557 (100 mg) | 2500 (1 mg) 2501 (10 mg) | 1312 (labels 25 µg Ab/reaction) 1310 (labels 100 µg Ab/reaction) |
| PE-iFluor® 594 | Blue/Green/Yellow | 495, 546, 566 | 606 | 2600 | 2584 | |
| PE-Texas Red | Blue/Green/Yellow | 495, 546, 566 | 615 | 2619 | 2583 | 1343 (labels 25 µg Ab/reaction) 1318 (labels 100 µg Ab/reaction) |
| PE-iFluor® 610 | Blue/Green/Yellow | 495, 546, 566 | 625 | 2700 | ||
| PE-iFluor® 647 | Blue/Green/Yellow | 495, 546, 566 | 666 | 2702 | 2577 | |
| PE-Cy5 | Blue/Green/Yellow | 495, 546, 566 | 666 | 2610 | 2580 | 1340 (labels 25 µg Ab/reaction) 1322 (labels 100 µg Ab/reaction) |
| PE-Cy5.5 | Blue/Green/Yellow | 495, 546, 566 | 671 | 2613 | 2581 | 1341 (labels 25 µg Ab/reaction) 1316 (labels 100 µg Ab/reaction) |
| PE-iFluor® 660 | Blue/Green/Yellow | 495, 546, 566 | 695 | 2602 | 2579 | |
| PE-iFluor® 700 | Blue/Green/Yellow | 495, 546, 566 | 708 | 2614 | 2585 | |
| PE-iFluor® 710 | Blue/Green/Yellow | 495, 546, 566 | 740 | 2615 |
Selecting Fluorophores for the Development of Multicolor Phenotypic Panels
In multicolor flow cytometry design, it is important to be familiar with the instrument's configuration that a particular phenotypic panel is being designed for, the cell lineage subpopulations, their expected antigen density, and the available antibody-conjugates and their properties. 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 in 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 is recommended to minimize interference. To minimize any signal spill-over, 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.
Three-laser configuration spectral flow cytometers, such as the Cytek Aurora, offer exceptional multi-parametric data for a wide array of applications. Markers and fluorochromes in a 24-color panel designed for identification of circulating cell subsets in human peripheral blood are summarized in the table below:
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 γδ | 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™ |
- Brilliant Violet™ is a trademark of Sirigen Group Ltd.
- eFluor® is a trademark of Thermo Fisher Scientific
Compatible Reagents for a 3-Laser Spectral Flow Cytometer
Many AAT Bioquest reagents can be combined in your spectral flow cytometry experiment. Use the following selection guide below to find available fluorescent antibody label options and functional reagents, such as DNA binding dyes, viability dyes, and ion indicators, for a spatially offset 3-laser spectral flow analyzer. Many of the listed fluorophores can be used in combination, however, there are still some incompatibilities.
Violet Laser Reagents
| Em Range (nm) | Best Dyes | Em max (nm) | Cell Cycle Dyes | Viability Dyes | Apoptosis Dyes | Cell Proliferation Dyes |
| 400-500 | iFluor® 405 | 427 | ||||
| mFluor™ Violet 420 | 427 | |||||
| mFluor™ Violet 450 | 445 | |||||
| PacBlue | 455 | |||||
| iFluor® 430 | 498 | |||||
| iFluor® 440 | 480 | |||||
| 500-600 | mFluor™ Violet 500 | 501 | ||||
| mFluor™ Violet 510 | 505 | |||||
| mFluor™ Violet 540 | 535 | |||||
| mFluor™ Violet 545 | 543 | |||||
| mFluor™ Violet 550 | 550 | |||||
| 600-700 | mFluor™ Violet 590 | 591 | ||||
| mFluor™ Violet 610 | 613 |