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 DEC

07. December 2021

What is Multispectral Cytometry?

What is multispectral flow cytometry?
Multispectral flow cytometry is an analytical technique that allows the measurement of fluorescence or Raman spectra from individual cells in suspension. Building on the technology used in conventional flow cytometry, multispectral cytometry deals with a spectral overlap — the effect produced when a detector receives a mixture of signals from multiple labels — by utilizing a collection of detectors/ channels in multichannel detector arrays. Crucially, the quantity of detectors is greater than the number of labels, with no one detector assigned to a specific label a priori.
Multispectral cytometry has risen in prominence in recent years due to its advantages over its predecessors and cross-disciplinary applications; expanding beyond the clinic to food science and engineering. Biomedical uses for multispectral cytometry is also an important emerging field, transforming techniques surrounding the preservation of tissue integrity.

What is multispectral tissue cytometry?
Multispectral tissue cytometry addresses the difficulty in resolving challenges of spectral overlap while conducting tissue cytometry – a microscopic equivalent of flow cytometry – on tissue and cell samples. In a conventional flow cytometer, only cells in suspension can be analyzed while tissue cytometry enables the preservation of the cells within the tissue context. The main advantage of using multispectral imaging technology in tissue cytometry is that the number of stained/acquired biomarkers can be drastically increased.

How does it work?
In multispectral tissue cytometry, the optical pathway is designed using a series of mirrors and filters that direct photons from different fluorophores to dedicated photomultiplier tubes.
However, due to imperfect filters and spectral overlap, perfect separation is almost unobtainable and fluorescence emitted from every fluorophore is liable to reach multiple detectors. These difficulties pose a unique challenge when translated to performing tissue examination and imaging with high numbers of markers that the capabilities of multispectral cytometry can solve.
This technology, in conjunction with advanced software capable of unmixing overlapping signals (= spectral unmixing) to calculate the fluorescent abundances, allow multispectral tissue cytometry to recognize and record every molecule's fluorescent spectrum as a spectral signature.
This translates as an increased light collection in multispectral tissue cytometry, which has been shown by Feher et al. to enhance sensitivity, improve the detection limit and boost resolution. These advantages of multispectral cytometry allow for improved imaging and processing in studies dealing with biological tissues and cellular samples that are inherently difficult to examine because of the nature of overlapping fluorescence signals.

Benefits and considerations of multispectral tissue cytometry
Advancements in multispectral tissue cytometry have allowed the tailoring of techniques toward the study of tissues, the inclusion of more photodetectors, and high-powered LEDs extending the capabilities of the technology. Innovative multispectral tissue cytometry products such as the TissueFAXS SPECTRA by TissueGnostics possess the ability to conduct multispectral imaging in tissue, expanding the number of markers for and the potential for biomedical research and industrial applications.
Concurrent expansion in the repertoire of fluorescent reagents, especially in the ultraviolet and near-infrared region, has made multi-parametric analyses possible, albeit increasing the complexity of experimentation. Ultimately, the improved resolution of fluorophores and the greater number of parameters available during experimentation means that more markers can be added to panels and the characterization of rare cell subsets is possible. Multispectral tissue cytometry is invaluable for research in the field of cancer immunotherapy, as well as serving a range of therapeutic areas.
Biologists and biotechnologists are increasingly incorporating multispectral cytometry into biological workflows to effectively conduct multi-parametric analysis and single-cell analysis while offering greater flexibility in reagent choices and the elimination of autofluorescence. With its impressive specifications, it is important to understand that effectively performing multispectral cytometry requires many careful considerations. Appropriate use of markers and antigen density should be factored into experimental design when defining cell populations, and errors visible after spectral unmixing, so-called spillover-spreading errors, should be remembered. Furthermore, any limitations inherent in the instrument used should be factored into experiment design, and proper handling of equipment is essential in reducing human error.

At TissueGnostics, we produce a range of innovative equipment for use in multispectral cytometry, if you would like to know more contact us today.

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References
Feher, K., et al. 2016. Multispectral flow cytometry: The consequences of increased light collection. Cytometry A.
https://doi.org/10.1002/cyto.a.22888 
Novo, D., et al. 2013. Generalized unmixing model for multispectral flow cytometry utilizing nonsquare compensation matrices. Cytometry A.
https://doi.org/10.1002/cyto.a.22272