Illuminating Immunology with Spectral Flow Cytometry
Published: October 30, 2023
Flow cytometry is a core technique in many immunology labs.. However, extensive immunophenotyping panels can result in overlapping emission peaks, time-consuming compensation and complex data analysis.
Spectral flow cytometry collects full, spectral fluorophore signatures, using unmixing algorithms to resolve overlapping emission peaks and produce accurate results. This removes the limits of panel size and reduces the need for compensation.
Explore how you can combine spectral cytometry with bright, stable and highly-efficient fluorophores to elevate your experiments.
Download this article to discover:
• How to improve signal resolution for rare cell subsets
• The best fluorescent dyes for highly accurate, reproducible data
• How to tackle the issue of autofluorescence
With Spectral Flow Cytometry
affect results and complicate data analysis. In addition,
tandem fluorophores can vary in batch quality and degrade
and dissociate over time, affecting the accuracy of signal
Since the first flow cytometers were developed for
and results (Drescher et al., 2021).
commercial use more than 40 years ago, flow cytometry
has become a cornerstone of many clinical and research
How can these challenges be overcome? The answer
laboratory workflows. Flow cytometry allows the
may lie in spectral flow cytometry – a rapidly growing,
determination of a cell’s size, granularity, and expressed
relatively new technology that captures the full emission
proteins by assessing its light-scattering properties. Hence,
spectra of fluorophores. Spectral flow cytometry allows
it is a powerful tool for the detailed analysis of complex
the highly precise determination of over 40 different
cell populations. As cells suspended in a saline solution
fluorophores, even with heavily overlapping emission
are passed in a single stream through a laser light source,
spectra and highly autofluorescent cells. Thus, this
visible light scattering provides information about the
technology promises to open the door to a new,
diameter (forward scatter – FSC) and the granularity (side
illuminated world of flow cytometry.
scatter – SSC), while the addition of fluorescent dyes or
What is spectral flow cytometry?
fluorophore-conjugated antibodies enables phenotypical
characterization and cell sorting (McKinnon, 2018).
The first iteration of spectral flow cytometry was
Flow cytometry is used comprehensively across multiple
developed in 1979, when researchers managed to extract
disciplines in a large range of applications, including cell
full emission spectra during flow cytometry analysis (Wade
cycle analysis, assessing RNA expression, cell sorting,
et al. 1979). However, it wasn’t until the early 2000s that
and absolute cell counting. However, the most common
spectral instruments with sufficient sensitivity and speed
application is immunophenotyping, i.e., identifying
immune cells in a mixed population by their surface
markers (McKinnon, 2018). Immunophenotyping assays
So, what is so special about spectral flow cytometry?
rely on the ability of modern cytometers to simultaneously
In conventional flow cytometry, one specific detector
measure increasing numbers of parameters. While
is used per fluorophore to collect emitted photons in
the first flow cytometers could only measure one or
the region exhibiting maximum emission, blocking or
two different fluorescent parameters, today around 30
reflecting all other wavelengths. In comparison, spectral
parameters can be analyzed simultaneously (Drescher et
flow cytometers disperse photons according to their
wavelength, which are then collected by an array of
detectors (Figure 1) (Nolan & Condello, 2013). Therefore,
For all the rapid developments in flow cytometry, challenges
the full spectral signature of each fluorophore is collected,
and limitations continue to plague researchers. As anyone
allowing researchers to resolve fluorescent dyes that have
who has ever designed a large antibody panel will know,
highly overlapping emission spectra, meaning that the
the more fluorophores there are in a panel and the closer
panel size isn’t limited by the number of channels on the
their emission peaks, the harder they are to differentiate.
instrument (Ramirez, 2022). Obviously, this creates an
This then demands complex compensation, which can
enormous amount of information that needs to be turned
often be miscalculated. Cellular autofluorescence can also
into usable data. Firstly, the spectra of all the different
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