Complex, high parameter flow cytometry panel design is now possible with spectral technology. But spectral offers advantages versus conventional even for routine, lower-parameter questions.
This guide breaks down the advantages of spectral flow cytometry for different experiment types and explores reasons to consider trying spectral technology.
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Overview of spectral and high parameter flow cytometry
Flow cytometry is a fundamental life science technique that allows researchers to analyze the physical and chemical characteristics of cells or particles as they flow through a laser beam. Each measured characteristic is referred to as a parameter.
Flow cytometry relies on fluorescence, where specific fluorophores are used to stain different cellular components. But as researchers push the boundaries of multi-parameter analysis, they are running into the limitations of conventional flow cytometry and inevitable fluorophore signal overlap.
Enter spectral flow cytometry, which can capture the full emission spectrum of every fluorophore using a wider range of detectors. Spectral flow technologies today can support high parameter flow cytometry with 20, 30, or even 40+ parameters of analysis.
The field of spectral flow cytometry is still relatively young and evolving quickly. As early adopters pioneer applications like discriminating pneumonia infections triggered by SARS-CoV-2 infection from those with other causes, a new wave of researchers is eager to dip their toes in the water and learn more. Some conventional flow users may still feel apprehensive, however, about the prospect of tackling a new and potentially more complex methodology.
The good news is that the transition may not be as daunting as it seems. Spectral panel design can be more straightforward than conventional in certain applications – including low-parameter – and provide other benefits that conventional flow cytometry cannot.
The challenge of panel design and spectral overlap in flow cytometry
Panel design in flow cytometry is the process of selecting fluorophores to accurately measure markers of interest. It is one of the greatest challenges that flow users face. In fact, flow cytometry typically involves far more time invested in planning and preparing an experiment than in executing it.
There are many puzzle pieces involved in panel design: biological considerations, instrument configuration, sample preparation methods, control design, experimental goals, data analysis, validation. It’s not necessarily a linear process, either. Researchers may enter the design process from different points in the workflow.
In the end, planning involves the same basic steps for both conventional and spectral flow. But a few key differences make a large impact.
Advantages of spectral flow cytometry for high parameter panel design
For high-parameter studies, the benefit of a spectral approach is clear. A spectral flow cytometer’s ability to record the entire emission spectrum of each fluorophore creates “spectral fingerprints” for clear differentiation of markers.
Instead of the compensation methods necessary to detangle fluorophore signals in conventional flow cytometry, spectral methods use algorithmic “unmixing.” In this way, resolution of fluorophores is possible even when using dyes with near-identical peak emissions and/or exploring more than 20 markers of interest. Conventional compensation matrices are particularly error-prone in high-parameter panels (>20 markers), which can lead to misinterpretation of dim or overlapping signals.
Spectral users may even find fluorophore and reagent selection to be easier than expected due to the rise of special, plug-and-play web tools that simulate panel optimization. These kinds of tools empower less experienced users to better understand spectral signatures and compatibility with less need for wet lab data.
Finally, spectral cytometry can have an advantage over conventional methods in optionally enabling the extraction of autofluorescence. When used with an unstained control tissue sample, autofluorescence extraction can improve signal clarity on runs.
Advantages of spectral for low parameter panel design
Surprisingly, spectral flow cytometry can offer advantages over conventional flow for routine, low-parameter experiments as well.
For one, the simplicity of spectral unmixing relative to conventional compensation holds just as true in low-parameter designs. With fewer markers and therefore single-color controls to prepare than in high-parameter assays, avoiding the more manual compensation process may be preferable. The spectral unmixing process is typically an automated, user-friendly operation performed by sophisticated software.
Dim or otherwise challenging dyes that are considered problematic for conventional instruments often work well in spectral panels, where the instrument is more capable of isolating accurate signatures.
Finally, performing low-parameter studies on a spectral flow cytometer can help advance your work in the long term – leaving the door open for expansion into more research parameters for the same experimental question.
| Spectral flow cytometry | Conventional flow cytometry | |
|---|---|---|
| Spectral detection | Multiple detectors capture full emission spectrum of fluorophore marker | Single detector filters narrow band of light from fluorophore marker |
| Data analysis & correction | Unmixing algorithm software separates spectral signatures; can subtract autofluorescence | Compensation process corrects for spillover between similar fluorophores |
| Parameters | 30+ | ~15-30 |
| Common applications | Large and complex immunology panels; low-parameter panels with overlapping fluorophores | Standard multicolor analysis with or without image analysis |
5 reasons to try spectral flow cytometry in your research
Whether you’re designing panels for high-parameter or low, spectral flow analysis is worth considering. To summarize some of the advantages that we’ve already discussed, spectral flow cytometry can offer:
- Flexibility in fluorophore selection and minimal spectral overlap risk
- Better resolution of autofluorescence
- Elimination of the traditional compensation process in lieu of software-driven algorithmic unmixing
- Exceptional multiplexing options in addition to quality low-parameter utility
- Timely experience in a scalable and quickly advancing method in life science
It’s a great time to jump into spectral with a multipurpose instrument
Truly, now is a great time to jump into understanding spectral flow cytometry and advancing your own knowledge in this nascent, innovative corner of cell and protein analysis.
Industry partners have also been opening the field up to a wider range of researchers, labs, and budgets. Tools like the Attune Xenith Flow Cytometer, which launched at the end of 2024 and into early 2025, are democratizing access by blending the advantages of conventional and spectral flow cytometry into a single instrument.
The Attune Xenith cytometer, as usual, offers one of the fastest run-times available for conventional flow cytometry (you can read more about the acoustic focusing technology behind its speed here) while including the 6 laser and 51 fluorescent channels needed to push the boundaries with spectral cytometry.
As more dyes, reagents, and advanced data analysis tools for high-parameter work emerge – particularly in parallel with exciting AI technology advances – spectral flow cytometry methods will only improve.
Investing time now in understanding spectral techniques, technologies, and datasets may pay off with dividends later. Today it is possible to invest in equipment that supports both spectral and conventional flow cytometry, bridging the gap between two important techniques that are here to stay in the field of cell analysis.
More flow cytometry resources
- Learn more about the new Invitrogen Attune Xenith Flow Cytometer with conventional and spectral flow capabilities
- Visit our Flow Cytometry Solutions Center
- Learn flow at your own pace at the Molecular Probes School of Fluorescence
- Explore expert opinions with the “Let’s Talk Flow” on-demand webinar series
- Learn more about spectral flow cytometry panel design or use our free, expert-led panel design service
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