Isolating subsurface fluorescence in macroscopic lifetime imaging via high-spatial-frequency structured 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 develop a modulation transfer function linking spatial frequency with signal penetration depth through comprehensive Monte Carlo eXtreme 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 imaging system. Further demonstrating its practical utility, we successfully applied HSF-FLI to preclinical drug delivery assessments employing Förster resonance energy transfer MFLI. The method was rigorously validated <i>in vivo</i> using mouse tumor xenograft models and cross-validated through <i>ex vivo</i> analyses. Overall, by integrating structure illumination techniques with physics-based depth modeling, HSF-FLI achieves precise depth-selective FLI. This advancement significantly enhances the accuracy, biological interpretation, and applicability of FLI, positioning HSF-FLI as a valuable tool for translational research.