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July 18th, 2025
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Université Laval
bioinformatics
biorxiv

Enhancing STED Microscopy via Fluorescence Lifetime Unmixing and Filtering in Two-Species SPLIT-STED

Deschenes, A.Open in Google Scholar•Ollier, A.Open in Google Scholar•Lafontaine, M.Open in Google Scholar•Michaud-Gagnon, A.Open in Google Scholar•Santiague, J.-G. S.Open in Google Scholar•Bilodeau, A.Open in Google Scholar•Gagne, C.Open in Google Scholar•De Koninck, P.Open in Google Scholar•Lavoie-Cardinal, F.Open in Google Scholar

Simultaneous super-resolution imaging of multiple fluorophores remains a major challenge in STimulated Emission Depletion (STED) microscopy due to spectral overlap of STED-compatible fluorophores. The combination of STED microscopy and Fluorescence Lifetime Imaging Microscopy (FLIM) offers a powerful alternative for super resolved, multiplexed imaging of biological samples but is hindered by lifetime convergence at high depletion powers. Here, we present an analysis method, two-species Separation of Photons by LIfetime Tuning (SPLIT)-STED, that uses a linear system of equations in phasor-based STED-FLIM to enhance both fluorophore unmixing and spatial resolution. It defines the fluorescence signal as a mixture of three lifetime components: the two target fluorophores and a short-lifetime contribution from undepleted peripheral fluorescence photons. Two-species SPLIT-STED disentangles overlapping lifetimes and selectively filters low-resolution signal. The method enables accurate unmixing of spectrally overlapping fluorophores and, by enhancing resolution through lifetime-based filtering, allows the use of lower depletion powers, thereby improving fluorescence lifetime separation.

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