Illuminating Immunology with Spectral Flow Cytometry

Spectral cytometry Illuminating Immunology With Spectral Flow Cytometry affect results and complicate data analysis. In addition, Introduction 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 were developed. 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 al., 2021). 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