Focused fluorescence excitation with time-reversed ultrasonically encoded light and imaging in thick scattering media

Abstract

Scattering dominates light propagation in biological tissue, and therefore restricts both resolution and penetration depth in optical imaging within thick tissue. As photons travel into the diffusive regime-typically 1 mm beneath human skin, their trajectories transition from ballistic to diffusive due to increased number of scattering events, which makes it impossible to focus, much less track, photon paths. Consequently, imaging methods that rely on controlled light illumination are ineffective in deep tissue. This problem has recently been addressed by a novel method capable of dynamically focusing light in thick scattering media via time reversal of ultrasonically encoded (TRUE) diffused light. Here, using photorefractive materials as phase conjugate mirrors, we show a direct visualization and dynamic control of optical focusing with this light delivery method, and demonstrate its application for focused fluorescence excitation and imaging in thick turbid media. These abilities are increasingly critical to understanding the dynamic interactions of light with biological matter and processes at different system levels, as well as their applications for biomedical diagnosis and therapy.

Keywords: Fluorescence imaging; Optical focusing; Optical imaging; Optical scattering; Phase conjugation; Photorefractive effect; Time-reversal; Ultrasound-modulation.

Figure 1. (color online) Schematic of focused fluorescence excitation in turbid media with TRUE light

(a) Holographic recording of ultrasound-modulated photons. (b) Phase conjugated copies of ultrasound-modulated photons travel “time-reversely” to the US focus and excite the fluorescent target. (c) The essential components of the experimental setup: BS, beam splitter; D, fluorescence detector (a CCD camera or an avalanche photodiode, APD, in this study); LPF, long-pass filter; OS_1-3, optical shutters; PCM, phase conjugation mirror; PD_1,2, photodiodes; R, reference beam; R*, reading beam; RL_1-3, relay lenses; S, incident sample beam; UT, ultrasound transducer; XYZ, system coordinates.

Figure 2. (color online) Direct visualization of TRUE optical focusing within thick turbid media

(a) Illustration of sample arrangement. A fluorescent bar was embedded centrally inside a transparent gel sandwiched between two scattering layers. The center of the US focus was aligned to intercept the center of the fluorescent bar in both the Y and Z directions. (b-d) CCD images of the fluorescence emission under the illuminations of (b) the incident sample beam (DC), (c) the phase conjugated beam of the unmodulated photons (TRDC), and (d) the tightly focused phase conjugated beam of the modulated photons (TRUE). (e) Intensity distributions of fluorescence signals excited by the DC, TRDC, and TRUE light along the white dashed lines in (b-d), respectively.

Figure 3. (color online) Fluorescence imaging of small objects embedded inside a thick turbid medium

(a) Illustration of sample arrangement. (b) Comparison of fluorescence images excited by the DC, TRDC, and TRUE light, respectively. (c) Comparison of DC, TRDC, and TRUE images based on the absorption contrast of 532 nm excitation light. The discrete symbols in (b) and (c) represent the experimental data, and the dashed curves represent Gaussian fits to the TRUE measurements.