Phitonex is now a part of Thermo Fisher Scientific. There is no change for our customers at this time to sales or service contacts or processes. We look forward to sharing more on the benefits of the acquisition for our customers soon. Learn more about: Thermo Fisher Scientific.

Above and Beyond 40 Colors

Sharing what is possible when you unlock high-resolution biology in flow cytometry with spectral clarity + what happens when you simultaneously push the envelope of ease-of-use, stability, and immunophenotyping.

From QDots to polymer dyes, hardware innovation in flow cytometry has been a response to a rapidly expanding dye portfolio. However, we have entered a new flow cytometric era in which there has been an inversion, and hardware has far outpaced dye technology. The rise of spectral flow cytometry suggests that there will be a seemingly endless expansion of parameters employed in a panel, thus eliminating previous constraints in panel design. Spectral cytometry takes advantage of off-target fluorescence to make use of a dye’s signature, rather than its fluorescence in a specific channel, relying on the underlying poor performance of currently available dyes to separate their signals.

The Spectral Clarity of Phitonex Labels Transform What is Possible

Taking a shared 35 color panel [1] we have used the 34 commercially available conjugated antibodies and incorporated our 6 new fluorescent labels. The spectral cleanliness of our platform, which allows us to design fluorescent labels that emit at targeted wavelengths with up to 70% less cross excitation than conventional tandem dyes, enabled us to plug-and-play these labels into this existing panel to increase the number of questions per cell (colors), and push what is both achievable and analyzable.

Our first 6 NovaFluor™ labels are excited uniquely by either blue or yellow-green lasers, which open up new spectra as compared to existing PE-tandem dyes. In addition, they:

bright and clean: limited cross-excitation means the best performing fluorescence and the clearest data

consistent and stable: leave them on a bench or in fixative, these labels are incredibly stable

tunable: the excitation, emission, and brightness are all controllable, moving dyes into the future

plug-and-play: “upgrade” your current instruments and use all current detectors off the B/YG to uncover more biology

discover more: stop letting dye performance get in the way of your analysis. Lower spillover means higher resolution.

Open for Innovation

We are sharing (1) our data under a Creative Commons License so they can serve as a resource for the field, (2) our observations about dye and hardware performance and (3) our supplemental information. We will update our online resources as we continue to analyze this data.


NOVAFLUOR
SPECTRAL CLARITY

Clean fluorescence brings higher parameters + cleaner data to every
flow cytometer.

Our technology platform enables us to answer a fundamental question – what if you could design a fluorescent label from scratch? We then leveraged this insight from our customers to create fluorescent labels built for biology, overcoming the myriad challenges posed by spectral spillover (i.e., fluorescent emission from one label that overlaps with one or more other labels) and cross-excitation (i.e., excitation of a label by multiple lasers).

NovaFluor labels are built using our proprietary Phiton™ technology, which leverages decades of innovation in the chemical modification of DNA [2-12], well understood oligo synthesis to ensure consistency, and DNA self-assembly to control fluorescent properties. (Figure 1).

Leveraging this fluorescence-by-design capability, NovaFluor spectra are engineered to avoid cross-excitation between laser lines (Figure 2), a common problem with conventional labels, while the emission profiles are tuned to avoid spectral spillover into undesired channels [13-14]. This enables us to maximize the detection capabilities of all current flow cytometers by cleanly “stacking” laser lines with high performance, spectrally clean labels.

FIGURE 1 | The Phiton Platform: Fluorophore-functionalized single-stranded DNA (ssDNA) is folded into a predefined structure (i.e., a Phiton). The platform can be conjugated (like current dyes) to antibodies (Ab), or other macromolecules, for specific downstream applications including flow cytometry.

FIGURE 2 | Diagram indicating that PE-Dazzle is excited by both blue and
yellow-green lasers, whereas similarly emitting NovaBlue™ 610 and NovaYellow™ 610 dyes are separable by the laser utilized for excitation. This is an example of how Phiton-based technology resolves a critical cross-excitation issue that is commonly observed with PE-tandem dyes.


PLUG-AND-PLAY PLEX

NovaYellow and NovaBlue labels deliver more insight, immediately.

Pushing the envelope should not mean more difficult data analysis or panel design, and we set out to prove this. Based on a previously shared multicolor panel of 35 colors [1], we started with the 34 commercially available conjugated antibodies and dropped in 6 additional NovaFluor-conjugated antibodies. Thanks to NovaFluor spectral clarity, performance, and easy conjugation, we were able to immediately plug and play 6 additional colors with limited impact on the current panel, thus pushing our ability to perform deep immunophenotyping without adding complexity in either panel design or data analysis.


40 COLOR PANEL

TABLE 1 | Specificities and fluorescent labels for the 40-color panel are shown above, based on the 35-color Cytek Aurora Panel. Human peripheral blood mononuclear cells (PBMCs) were stained as described in the Methods and Materials.


FIGURE 3 | Blue-excited dye emission spectra (top) and Yellow-Green-excited dye emission spectra (bottom). Figure was generated using FPBase [15]. Please note that BB515 is missing from this top plot, as we were unable to unmix it from FITC.


Data

We have shared the unmixed and raw data, as well as our analysis, on our Github page as well as Flow Repository.


Download this whitepaper to learn how Phitonex Labels plug-and-play into current flow cytometry experiments and push the envelope of immunophenotyping to 40 colors:

  • Discover more, now: spectral clarity means that NovaFluors can “upgrade” your current panels and uncover more biology, using current instruments
  • Fluorescent labels built for biology: NovaFluor stability, consistency, and our qc-on-cells commitment set a new standard for your reagents
  • High-parameter does not have to mean high stress: lower spillover means higher resolution of populations and easier, faster data analysis and interpretation

To learn more about how Phitonex can bring high resolution biology to your lab, please fill out the form below.

    Any information collected on this website will be kept confidential and will not be sold, rented, loaned, or otherwise disclosed.


    PE/Dazzle™ is the trademarks and property of BioLegend,Inc. Brilliant Violet™ is a trademark of Sirigen Group Ltd. BD Horizon™, Brilliant Blue (BB), Brilliant Violet (BV), and Brilliant UV (BUV) are trademarks of BD Biosciences. Alexa Fluor®, eFluor®, LIVE/DEAD™, Qdot™, and Super Bright are trademarks of Thermo Fisher Scientific.

    CF® is a registered trademark of Biotium. Cy® and CyDye® are registered trademarks of GE Healthcare Allophycocyanin (APC) conjugates: US Patent No. 5,714,386 PE-Cy7: US Patent Number 4,542,104. NovaBlue, NovaYellow are trademarks of Phitonex, Inc. Trademarks are the property of their respective owners.


    References

    1. “Cytek Aurora: Say Hello to a New Reality.” Cytek Bioscience, Inc. 2019.
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    3. C. LaBoda, C. Dwyer, A. R. Lebeck. “Exploiting Dark Fluorophore States to Implement Resonance Energy Transfer Pre-Charge Logic”, IEEE Micro, 37(4): 52-62, 2017.
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    14. https://www.sonybiotechnology.com/us/
    15. Lambert, TJ (2019) FPbase: a community-editable fluorescent protein database. Nature Methods. 16, 277–278. doi: 10.1038/s41592-019-0352-8.