Isolating Subsurface Fluorescence in Macroscopic Lifetime Imaging via High-Spatial-Frequency Illumination

Macroscopic Fluorescence Lifetime Imaging (MFLI) has emerged as a robust, non-invasive imaging technique offering quantitative insights into physiological and molecular processes within live tissues, independent of fluorophore concentration, excitation intensity, or signal attenuation. However, a key limitation is the inability to accurately determine the depth at which fluorescence signals originate, potentially compromising biological interpretation due to ambiguous localization. In this study, we introduce High Spatial Frequency-Fluorescence Lifetime Imaging (HSF-FLI), an innovative optical correction methodology designed to effectively eliminate surface signal bias, such as those arising from skin in preclinical imaging, without requiring chemical clearing agents. We developed a modulation transfer function (MTF) linking spatial frequency with signal penetration depth through comprehensive Monte Carlo eXtreme (MCX) simulations. Utilizing structured, three-phase sinusoidal illumination, fluorescence signals were accurately decomposed into distinct surface and subsurface components. Experimental validation was performed using agar-based capillary phantoms and a time-gated Intensified Charged Coupled Device coupled with a Digital Micromirror Device (ICCD–DMD) imaging system. Further demonstrating practical utility, we successfully applied HSF-FLI in preclinical drug delivery assessments employing F¨orster Resonance Energy Transfer (MFLI-FRET). The method was rigorously validated in vivo using mouse tumor xenograft models and cross-validated through ex vivo analyses. Overall, by integrating structure illumination techniques with physics-based depth modeling, HSF-FLI achieves precise depth-selective fluorescence lifetime imaging. This advancement significantly enhances the accuracy, biological interpretation, and applicability of fluorescence lifetime imaging, positioning HSF-FLI as a valuable tool for translational research.