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

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 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 4σ, 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. Ultimately, the primary value of MSSR-based spatial refinement lies in leveraging improved spatial resolution to effectively isolate the temporal profiles of mixed fluorophore populations preserving the integrity of FLIM measurements.