Expansion of Fluorophores for Spectral Flow Cytometry

In 2004, Perfetto and Roederer published the first paper detailing a 17-color flow cytometry experiment by incorporating a relatively new octagon emission array off the violet 405nm laser (1). The additional laser and 8 additional channels for violet excited fluorophores facilitated the use of 6 Qdot Nanocrystal conjugates in the first big reach towards high parameter flow cytometry assays. In 2012, BioLegend released the first Brilliant Violet conjugates to make 18 color flow cytometry more accessible to researchers by offering an alternative to QDots. However, the big turning point was the release of the Cytek Aurora in 2017.  A mere 7 years later, the first 50 color assays are being published on the BD Discovery S8 system (2). The Cytek Aurora, Sony ID7000, and BD Discovery S8 are all poised to take flow cytometric assays into the new age of big data and the untangling of complex biological relationships. As the capabilities of instrumentation have grown, reagents must rise to the challenge with direct antibody conjugates with fluorophores developed with an eye to spectral diversity and spectral stability.

Commercial reagent manufacturers face several challenges to create fluorophores that improve upon current standards, including:

• Having discrete excitation by a single laser to reduce cross-beam excitation 
• Tunable emission peaks to fill the gaps between conventional emission peaks
• Narrow emission profiles to reduce spectral spillover into neighboring channels 
• Efficient quenching of the donor molecule in a tandem dye, which limits the emission back into the donor channel
• Bright or tunable emission brightness to match the abundance level of antigens
• Minimal non-specific binding of fluorophores
• Stability of fluorophores to light, temperature and various fixative solutions

In this article, we highlight reagents that are paving the way to meet the needs of spectral cytometry.

Fluorophores Made of Simple Organic Chemical Structures

In a quick response to the increasing demand for a greater repertoire of fluorescent emission profiles, researchers first looked to simple existing organic fluorophores that could emit light at wavelengths between those of traditional fluorophores, like PE, APC, PerCP tandems and the Brilliant Violet and UV dye families. These dyes could be quickly and easily conjugated to primary antibodies and validated in multicolor combinations. BioLegend released a number of Spark fluorophores, such as Spark Blue 550 and Spark NIR 685, validated for applications run on the Aurora. There are now around 13 fluorescent dyes in this group available for use.

Improvements and Diversification of PE, APC and PerCP tandem conjugates

Another important goal of reagent development at the beginning of the spectral cytometry movement was to make PE, APC and PerCP tandems more stable to light, temperature, and fixatives. This is crucial to ensure the reliability of the spectral emission profile, the standard needed to apply unmixing accurately. If the shift of emission profiles is not reflected in unmixing controls, the unmixing becomes inaccurate. Although tandem inefficiencies and instabilities have always been an issue in flow cytometry, this problem was exacerbated in assays over 17 colors. The fluorescent tandems listed below were designed to be superior, more photostable replacements to the traditional protein-based tandem dyes, like : PE-Texas Red/ECD, PerCP-Cy5.5 and APC-Cy7.

In addition to stabilizing common tandems to resist degradation by light, temperature, and fixative, researchers have expanded the emission range by pairing novel acceptor dyes with traditional donor dyes. In response to the increase in efficiency of APDs in the NIR, BioLegend developed tandem dyes with emission peaks greater than 790 nm, such as PE-Fire 810, PerCP-Fire 780, PerCP-Fire 806 and APC-Fire 810. In addition, PE-Fire 744 fills in a large gap in the emission spectrum between existing PE tandems, like PE-Cy5.5 and PE-Cy7.

Brilliant Violet and Brilliant UV fluorescent Polymer Family

The development of the new class of fluorescent polymers, called the Brilliant family, was instrumental in enabling universal access to high-parameter assays. Prior to this development, researchers had to conjugate their own purified antibodies or order custom conjugations of QDot nanocrystals, which only had a guaranteed shelf life of six-months, resulting in limited commercial availability of direct conjugates.

 When the Brilliant Violet family was first developed as a collaboration between BioLegend and Sirigen (3), the emission peaks of these dyes were designed to overlap with existing QDot emission channels excited by the 405nm violet laser. This allowed for the commercial availability of direct conjugates to rapidly scale up to meet demand. 

Shortly after, BD Biosciences developed the Brilliant UV polymers. Now, direct conjugates of the Brilliant Violet dyes are commercially sold by multiple suppliers, with BD Biosciences and Thermo Fisher offering direct conjugates of the Brilliant UV dyes. However, there are no commercial kits available for researchers to conjugate these dyes themselves. 

StarBright Fluorescent Polymer Dots (pDots)

Meanwhile, Daniel Chiu of University of Washington was working on a similar polymer structure with a very different tertiary conformation (4). Rather than a linear polymer, Dr. Chiu created the polymer dot, which has been subsequently commercialized by Bio-Rad under the brand name StarBright. This class of dyes offers an alternative to the Brilliant polymer family.

The developers of the StarBright fluorophores had the foresight to spectrally offset the emission peaks for some existing fluorophores. This reduced the total spectral overlap with existing reagents, making these dyes more compatible with reagents developed up to this point. The StarBright dyes were also designed to more efficiently quench the donor fluorophore, resulting in lower complexity and spreading error related to spectral spillover. 

NovaFluor and KiraVia DNA Backbone Fluorophores

As the demand for polymerized fluorophores continued growing, researchers explored using a unique polymerizing backbone to fill the void. In 2013, research within chemical biology was already underway to substitute nucleotides with fluorophores from labs, like that of Eric Kool at Stanford (5).

DNA is nature’s original polymer that can self-assemble – its synthesis can be controlled for sequence, site-specific substitution of fluorescent nucleosides. Two new classes of fluorophores were developed with polymeric nucleotide backbones – the NovaFluors, originally developed by Phitonex and now supplied by Thermo Fisher, and the KiraVia fluorophores, developed by Sony and BioLegend. 

A key advantage of these fluorophores over the existing fluorescent polymerizing structures is their small size and the ability to finely tune both excitation and emission wavelengths, providing a cornucopia of spectral choices. However, both fluorophores from DNA or proteins from cyanobacteria can inadvertently bind to immune cells that scavenge for naturally occurring elements. It is therefore imperative to use monocyte blocking solutions provided by the manufacturer when applying these dyes to prevent non-specific binding. 

VioBright, RealBlue and RealYellow Multimers

Besides the structure mentioned previously, there are additional methods of multimer creation. Scientists at Miltenyi published a wonderful paper detailing current methods (6). Branded as “VioBright” by Miltenyi, these multimer dyes emit with very high brightness compared to other fluorescent polymers, despite the “Vio” name not referring to a violet laser dye specifically..

Within the last year, the RealBlue and RealYellow fluorescent multimers were released by BD Biosciences. While their exact chemical structure is unknown, they adhere to the previously mentioned standards for “good” fluorophore development. RealBlue, excited at 488nm, and RealYellow, excited at 561nm, have minimal cross-beam excitation by the unintended laser. This helps to reduce the total spectral complexity and spillover spreading error substantially. As fluorescent labeling continues advancing, we will likely see an expanding portfolio of these multimer dye products in the coming years.

References

1. Perfetto SP, Chattopadhyay PK, Roederer M. Seventeen-colour flow cytometry: unravelling the immune system. Nat Rev Immunol. 2004 Aug;4(8):648-55.

2. Konecny AJ, Mage P, Tyznik AJ, Prlic M, Mair F. 50-color phenotyping of the human immune system with in-depth assessment of T cells and dendritic cells. bioRxiv [Preprint]. 2023 Dec 15:2023.12.14.571745.

3. Chattopadhyay PK, Gaylord B, Palmer A, Jiang N, Raven MA, Lewis G, Reuter MA, Nur-ur Rahman AK, Price DA, Betts MR, Roederer M. Brilliant violet fluorophores: a new class of ultrabright fluorescent compounds for immunofluorescence experiments. Cytometry A. 2012 Jun; 81(6):456-66.

4. Wu C, Chiu DT. Highly fluorescent semiconducting polymer dots for biology and medicine. Angew Chem Int Ed Engl. 2013 Mar 11;52(11):3086-109. doi: 10.1002/anie.201205133.

5. Teo YN, Kool ET. DNA-multichromophore systems. Chem Rev. 2012 Jul 11;112(7):4221-45.

6. T. Reiber, O. Zavoiura, C. Dose, D. A. Yushchenko, Eur. J. Org. Chem. 2021, 2021, 2817.