Depth-Resolved Macroscopic Fluorescence Lifetime Imaging via High-Spatial-Frequency Structured Illumination.

SIGNIFICANCE: Macroscopic Fluorescence Lifetime Imaging (MFLI) is a powerful, non-invasive imaging modality that offers robust, physiologically relevant contrast largely independent of fluorophore concentration, excitation intensity, and tissue signal attenuation. However, accurately determining the depth of signal origin remains challenging, potentially leading to ambiguity in biological interpretation. Here, we present a novel optical correction method that effectively eliminates surface signal bias, such as that from skin in preclinical imaging, without the need for chemical clearance. This advancement supports the robust applicability of MFLI in translational research. AIM: Establishment of a High Spatial Frequency-Fluorescence Lifetime Imaging (HSF-FLI) framework to selectively isolate subsurface fluorescence (deeper signals) from surface fluorescence, while preserving the accuracy of lifetime estimation. APPROACH: A modulation transfer function (MTF) that relates spatial frequency to penetration depth was derived using Monte Carlo eXtreme (MCX) simulations (for physics-based modeling) and validated with agar-based capillary phantoms on a time-gated ICCD-DMD system. Depth-independent fluorescence was decomposed into surface and subsurface components through structured three-phase sinusoidal illumination, and nonlinear least squares fitting was applied to recover lifetime or lifetime based parameters maps. HSF-FLI was demonstrated in vivo in mouse models bearing tumor xenogratfs and was cross validated with ex vivo measurements. RESULTS: We extensively characterized the performance of High Spatial Frequency-Fluorescence Lifetime Imaging (HSF-FLI) through simulations and tissue-mimicking phantoms. The approach was further validated in vivo by assessing drug delivery in preclinical models using MFLI-FRET (Förster Resonance Energy Transfer). CONCLUSION: By coupling structured illumination with physics-based depth modeling, HSF-FLI delivers accurate, depth-selective lifetime readouts, setting the stage for robust and fast FLI implementation in translational studies.