High-spatial-frequency structured illumination enhances fluorescence lifetime specificity in scattering media

Macroscopic Fluorescence Lifetime Imaging (MFLI) enables concentration-independent contrast imaging in biological samples but is often compromised by surface fluorescence bias. To address this, we developed High Spatial Frequency Fluorescence Lifetime Imaging (HSF-FLI), which integrates three-phase structured illumination to selectively isolate subsurface signals while preserving lifetime specificity. Validation in Monte Carlo based simulations, tissue-mimicking phantoms and in vivo murine tumor models using MFLI-FRET confirmed effective suppression of surface artifact, achieved without the need for chemical clearing. HSF-FLI thus enables rapid, depth-selective lifetime imaging, markedly enhancing the specificity and translational potential of preclinical fluorescence imaging.