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bioRxiv · 10.64898/2026.03.17.712453

PSF-Driven Spatio-Temporal Blending in Fluorescence Lifetime Imaging Microscopy and Its Mitigation via Mean-Shift Super-Resolution-Based Masking.

Gonzalez-Gutierrez, M.·Vazquez-Enciso, D. M.·Mateos, N.·Hwang, W.·Torres-Garcia, E.·Hernandez, H. O.·Chacko, J. V.·Coto Hernandez, I.·Loza-Alvarez, P.·Wood, C.·Guerrero, A.
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Abstract

Fluorescence Lifetime Imaging Microscopy (FLIM) enables quantitative mapping of molecular environments in living systems with high biochemical specificity. However, spatial overlap dictated by the diffraction-limited point spread function (PSF) causes a mixing of temporal signals: photons from neighboring emitters collected within the same pixel yield composite decay profiles, generating apparent intermediate lifetimes that can be mistaken for variations in the local molecular environment. We introduce a workflow that applies Mean-Shift Super-Resolution (MSSR) to raw intensity data to generate intensity-derived spatial masks prior to phasor-based lifetime analysis. The method is computationally efficient and preserves decay kinetics because it operates on intensity-derived spatial information rather than modifying temporal data. In U2OS cells labeled with spectrally-overlapping fluorophores, phasor analysis reveals an intermediate lifetime population localized at PSF-overlap interfaces, consistent with optical mixing rather than intrinsic lifetime heterogeneity. MSSR-derived masking suppressed this mixed population while preserving stable phasor cluster centers -i.e. the distribution of similar phasor coordinates in the phasor plane- for each fluorophore. Simulations of strictly monoexponential fluorescence decay emitters further show that blended lifetime decay profiles are present at separations up to 4{sigma} and becomes maximal near [~]1.6{sigma}, indicating that conventional spatial resolution criteria can underestimate lifetime cross-talk. Application of this workflow to three-component FLIM showed also a reduced overlap of pixel distributions in phasor plots while maintaining distinct lifetime signatures. Overall, MSSR-based spatial refinement provides an accessible strategy to improve the spatial resolution while maintaining accuracy of FLIM measurements.

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Gonzalez-Gutierrez, M., Vazquez-Enciso, D. M., Mateos, N., Hwang, W., Torres-Garcia, E., Hernandez, H. O., Chacko, J. V., Coto Hernandez, I., Loza-Alvarez, P., Wood, C., Guerrero, A.. 2026-03-18. PSF-Driven Spatio-Temporal Blending in Fluorescence Lifetime Imaging Microscopy and Its Mitigation via Mean-Shift Super-Resolution-Based Masking.. https://doi.org/10.64898/2026.03.17.712453

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