Acousto-optic volumetric gating for reflection-mode deep optical imaging within a scattering medium

The imaging depth of deep-tissue optical microscopy is governed by the performance of the gating operation that suppresses the multiply scattered waves obscuring the ballistic waves. Although various gating operations based on confocal, time-resolved/coherence-gated, and polarization-selective detections have proven to be effective, each has its own limitation; certain types of multiply scattered waves can bypass the gating. Here, we propose a method, volumetric gating, that introduces ultrasound focus to confocal reflectance imaging to suppress the multiply scattered waves traveling outside the ultrasonic focal volume. The volumetric gating axially rejects the multiply scattered wave traveling to a depth shallower than the object plane while suppressing the deeper penetrating portion that travels across the object plane outside the transversal extent of the ultrasonic focus of 30\({\times}\)90\( {\mu}m^2\). These joint gating actions along the axial and lateral directions attenuate the multiply scattered waves by a factor of 1/1000 or smaller, thereby extending the imaging depth to 12.1 times the scattering mean free path while maintaining the diffraction-limited resolution of 1.5 \({\mu}\)m. We demonstrated an increase in the imaging depth and contrast for internal tissue imaging of mouse colon and small intestine through their outer walls. We further developed theoretical and experimental frameworks to characterize the axial distribution of light trajectories inside scattering media. The volumetric gating will serve as an important addition to deep-tissue imaging modalities and a useful tool for studying wave propagation in scattering media.