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

Deschenes, A.; Ollier, A.; Lafontaine, M.; Michaud-Gagnon, A.; Santiague, J.-G. S.; Bilodeau, A.; Gagne, C.; De Koninck, P.; Lavoie-Cardinal, F.

doi:10.1101/2025.07.17.661952

bioRxiv · 10.1101/2025.07.17.661952

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

Deschenes, A.·Ollier, A.·Lafontaine, M.·Michaud-Gagnon, A.·Santiague, J.-G. S.·Bilodeau, A.·Gagne, C.·De Koninck, P.·Lavoie-Cardinal, F.

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Abstract

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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Deschenes, A., Ollier, A., Lafontaine, M., Michaud-Gagnon, A., Santiague, J.-G. S., Bilodeau, A., Gagne, C., De Koninck, P., Lavoie-Cardinal, F.. 2025-07-18. Enhancing STED Microscopy via Fluorescence Lifetime Unmixing and Filtering in Two-Species SPLIT-STED. https://doi.org/10.1101/2025.07.17.661952

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