PSF-Driven Spatio-Temporal 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 stable phasor cluster centers - <i>i.e</i> . 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 and becomes maximal near ∼1.6σ, 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.