Isolating subsurface fluorescence in macroscopic lifetime imaging via high-spatial-frequency structured illumination.

Area of Science:

• Biomedical Optics
• Molecular Imaging
• Preclinical Research

Background:

• Macroscopic fluorescence lifetime imaging (MFLI) provides non-invasive, quantitative physiological insights.
• A major limitation of MFLI is the inability to determine fluorescence signal origin depth, hindering accurate localization.
• Surface signal bias, particularly from skin, complicates interpretation in preclinical MFLI.

Purpose of the Study:

• Introduce high spatial frequency-fluorescence lifetime imaging (HSF-FLI) to address depth localization challenges in MFLI.
• Develop an optical correction methodology to eliminate surface signal bias without chemical clearing.
• Enable precise depth-selective fluorescence lifetime imaging for improved biological interpretation.

Main Methods:

• Developed a modulation transfer function linking spatial frequency and signal penetration depth using Monte Carlo simulations.
• Employed structured, three-phase sinusoidal illumination to decompose fluorescence signals into surface and subsurface components.
• Validated HSF-FLI using agar capillary phantoms, time-gated intensified CCD, and digital micromirror devices.

Main Results:

• HSF-FLI successfully eliminated surface signal bias, enabling accurate depth localization of fluorescence.
• Demonstrated practical utility in preclinical drug delivery assessments using Förster resonance energy transfer MFLI.
• Rigorous in vivo validation in mouse tumor xenografts and ex vivo cross-validation confirmed method robustness.

Conclusions:

• HSF-FLI integrates structured illumination with physics-based depth modeling for precise depth-selective FLI.
• This advancement significantly improves the accuracy and biological interpretability of fluorescence lifetime imaging.
• HSF-FLI is positioned as a valuable tool for translational research, enhancing preclinical imaging capabilities.