Ratio-preserving super-resolution FLIM for quantitative live-cell lifetime imaging

Li, C.; Li, L.; Cheng, Y.; Lu, W.; Gou, W.; Li, H.; Zou, Q.; Saimi, D.; Chen, Z.; Gu, W.; Wang, Y.; Guo, C.; Liu, B.; Huang, X.; Fan, J.; Chen, L.

doi:10.64898/2026.10.25.761648

bioRxiv · 10.64898/2026.10.25.761648

Ratio-preserving super-resolution FLIM for quantitative live-cell lifetime imaging

Li, C.·Li, L.·Cheng, Y.·Lu, W.·Gou, W.·Li, H.·Zou, Q.·Saimi, D.·Chen, Z.·Gu, W.·Wang, Y.·Guo, C.·Liu, B.·Huang, X.·Fan, J.·Chen, L.

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Abstract

Super-resolution (SR) fluorescence lifetime imaging promises nanoscale maps of molecular environment and function, but photon-limited acquisition and computational reconstruction can distort lifetime estimates, particularly for ratio-derived frequency-domain measurements under SR conditions. Here we introduce structured-illumination frequency-domain FLIM (SIM-fdFLIM), an SR lifetime imaging platform that combines commercial dual-tap fdFLIM detection with ratio-preserving reconstruction. SIM-fdFLIM uses direct-current (DC)-stabilized lifetime estimation to improve reconstruction robustness under low signal-to-noise conditions, followed by ratio-domain integrated variational estimation (RIVE) to correct channel misregistration and suppress noise amplification after SR reconstruction. The method achieves ~100 nm spatial resolution in lifetime maps and enables live-cell imaging at 2.8 Hz over a 56 x 56 {micro}m2 field of view. Across representative fluorophore standards, RIVE reduces lifetime standard deviation by approximately threefold relative to non-RIVE SIM-fdFLIM while preserving mean lifetime agreement with calibrated widefield fdFLIM. In living cells, SIM-fdFLIM resolves mitochondrial cristae-scale lifetime heterogeneity, mitochondrion-to-mitochondrion functional dynamics, nanoscale lipid-environment variations and perturbation-sensitive organelle-contact signatures. These results establish SIM-fdFLIM as a ratio-preserving platform for quantitative SR lifetime imaging, extending live-cell FLIM into a regime that combines nanoscale spatial resolution, Hz-rate acquisition, and preserved lifetime fidelity.

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Li, C., Li, L., Cheng, Y., Lu, W., Gou, W., Li, H., Zou, Q., Saimi, D., Chen, Z., Gu, W., Wang, Y., Guo, C., Liu, B., Huang, X., Fan, J., Chen, L.. 2026-10-28. Ratio-preserving super-resolution FLIM for quantitative live-cell lifetime imaging. https://doi.org/10.64898/2026.10.25.761648

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