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

Area of Science:

• Biophysics
• Microscopy techniques
• Cellular imaging

Background:

• Fluorescence Lifetime Imaging Microscopy (FLIM) offers quantitative molecular mapping in living systems.
• Diffraction-limited point spread function (PSF) in FLIM causes spatial overlap, mixing temporal signals and creating apparent intermediate lifetimes.
• This optical mixing can be misinterpreted as genuine changes in the molecular environment.

Purpose of the Study:

• To introduce a novel workflow combining Mean-Shift Super-Resolution (MSSR) with FLIM for improved spatial resolution.
• To differentiate true molecular environment variations from artifacts caused by optical mixing.
• To enhance the accuracy and reliability of FLIM measurements.

Main Methods:

• Applied Mean-Shift Super-Resolution (MSSR) to raw intensity data to generate spatial masks.
• Utilized intensity-derived spatial masks prior to phasor-based lifetime analysis.
• Validated the workflow using U2OS cells with spectrally-overlapping fluorophores and through simulations.

Main Results:

• MSSR-derived masking effectively suppressed intermediate lifetime populations at PSF-overlap interfaces.
• The method preserved stable phasor cluster centers for individual fluorophores.
• Simulations confirmed that optical mixing artifacts extend beyond conventional spatial resolution limits.
• Reduced overlap in phasor plots was observed in three-component FLIM analyses.

Conclusions:

• MSSR-based spatial refinement is an efficient strategy to improve FLIM's spatial resolution.
• The workflow accurately distinguishes optical mixing artifacts from intrinsic lifetime heterogeneity.
• This approach enhances the biochemical specificity and quantitative accuracy of FLIM measurements in complex biological samples.