BPS2026 – Phasor-based analysis in spectral flow cytometry enabling high-throughput single-cell LAURDAN spectroscopy

The Phasor plot has become a powerful approach for the fit-free analysis of fluorescence spectra, allowing intuitive and unbiased interpretation of complex emission signals. While it has been widely used in hyperspectral microscopy, its potential in spectral flow cytometry has not yet been explored. Here, we introduce an implementation of phasor analysis in spectral flow cytometry and compare its performance to hyperspectral microscopy using the membrane probe LAURDAN. We applied phasor transformation to LAURADN fluorescence from multilamellar vesicles composed of DOPC and DPPC with cholesterol, as well as to living cells subjected to cholesterol modulation. Both cytometry and microscopy successfully identified the expected differences in membrane order, identifying increased phasor phases for membrane fluidity while shorter modulation, by cholesterol increase. Notably, spectral flow cytometry yielded tighter phasor distributions compared to microscopy, leading to less data spread because it averages information across the entire cell, though it sacrifices spatial resolution. This method allows for quantification of fluidity and cholesterol effects in high-throughput single-cell studies, providing enormous statistical power that is difficult to achieve with HSI. Spectral flow cytometry's compatibility with multiparametric measurements further enables integration with established flow cytometry assays, allowing for the direct correlation of spectral properties with immunophenotyping or functional readouts. Together, these results demonstrate that spectral flow cytometry enhances the high-throughput capabilities of HSI and phasor analysis, establishing a robust platform for quantitative single-cell spectroscopy. Phasor-based spectral flow cytometry will open new avenues for studying membrane organization, cellular metabolism, and drug responses, providing a scalable alternative to microscopy for applications requiring both sensitivity and throughput.