Abstract Macroscopic Fluorescence Lifetime Imaging (MFLI) has emerged as a robust, non-invasiveimaging technique offering quantitative insights into physiological and molecular processeswithin live tissues, independent of fluorophore concentration, excitation intensity, or signalattenuation. However, a key limitation is the inability to accurately determine the depthat which fluorescence signals originate, potentially compromising biological interpretationdue to ambiguous localization. In this study, we introduce High SpatialFrequency-Fluorescence Lifetime Imaging (HSF-FLI), an innovative optical correctionmethodology designed to effectively eliminate surface signal bias, such as those arisingfrom skin in preclinical imaging, without requiring chemical clearing agents. We developeda modulation transfer function (MTF) linking spatial frequency with signal penetrationdepth through comprehensive Monte Carlo eXtreme (MCX) simulations. Utilizingstructured, three-phase sinusoidal illumination, fluorescence signals were accuratelydecomposed into distinct surface and subsurface components. Experimental validation wasperformed using agar-based capillary phantoms and a time-gated Intensified ChargedCoupled Device coupled with a Digital Micromirror Device (ICCD–DMD) imaging system.Further demonstrating practical utility, we successfully applied HSF-FLI in preclinicaldrug delivery assessments employing F¨orster Resonance Energy Transfer (MFLI-FRET).The method was rigorously validated in vivo using mouse tumor xenograft models andcross-validated through ex vivo analyses. Overall, by integrating structure illuminationtechniques with physics-based depth modeling, HSF-FLI achieves precise depth-selectivefluorescence lifetime imaging. This advancement significantly enhances the accuracy,biological interpretation, and applicability of fluorescence lifetime imaging, positioningHSF-FLI as a valuable tool for translational research.
Yuan et al. (Tue,) studied this question.