Having selected proper light waves, researchers at the Center for Molecular Spectroscopy and Dynamics, within the Institute for Basic Science (IBS) have demonstrated a more than 10-fold improvement of light energy delivery to targets that are too deeply embedded to visualize with current optical imaging. Able to picture through a young mouse skull in the laboratory, this non-invasive technique does not cause any damage to tissues and does not need injections of fluorescent molecules to label the target. Published in Nature Photonics, this work might lay a foundation for in vivo experiments that use light in biomedical imaging, optogenetics, tumor treatment, and for recharging implanted medical devices.

Entering a complex and opaque environment like a human or animal body, light trajectory is deviated by all the components it finds on its way. For this reason, only a small fraction of light waves propagating inside biological tissues can actually reach the desired target, while the majority is scattered and randomly diffused. This multiple wave scattering hinders most applications of optical imaging for deep biological samples. IBS scientists found a strategy to maximize the intensity of waves that interact with the target over the waves that do not, which depends on the time it takes for the waves to travel to the target and be reflected back.

▲ Figure 1: Scheme of scattered light waves inside an inhomogeneous and opaque environment. IBS scientists are able to focus on the scattered waves that have interacted with the target and maximize their intensity. A body’s complex environment inhibits most of the light waves from reaching the target (green waves), whereas information about the target is presented in the red and blue waves. As target depth increases, the single-scattered signal (red) becomes negligibly small, because it is more likely that the waves will get bounced around by other biological molecules in their path (blue). Therefore, the goal of this study was to selectively control and enhance the blue waves over the green ones.

▲ Figure 2: Selecting the right waves. a) The goal of this study is to enhance the intensity of wave (3) over (1), (2) and (4), as (3) is the only wave which hit the target and thus contains information about it. b) IBS scientists took advantage of the different times it takes for the waves to be refracted back. Thanks to this technique, the wave which hits the target (wave 3) can stand out from the other waves. The final images show simulations with untargeted light signals (c) or targeted waves, equivalent to wave 3 (d). Compared to c), the waves in d) are less diffused laterally and reduce their intensity with increasing depth.

After having verified that theoretical predictions and experiments matched, IBS scientists applied the technique to a 340 μm-thick young mouse skull. They placed either one or ten silver disks below the skull as targets, and immersed them in a biological serum that simulates conditions of living tissue. The new technique, which registers only the waves that reached the target, worked better than collective accumulation of single scattering (CASS) microscopy.

▲ Figure 3: Peering through the skull. a) Either one or ten silver disks of 10 and 3 μm diameter, respectively, were placed below an approximately 340 μm-thick mouse skull and immersed in serum (PBS). b) The intensity of returning waves increased at a travel time of 3.2 picoseconds. c-d) Both targets were completely invisible to the collective accumulation of single scattering (CASS) optical imaging technique. e-f) Images of the transmitted waves using untargeted light signals, and g-h) images obtained with this new technique focusing on waves with travel time of 3.2 picoseconds.

“Optical imaging can generally work at the depth of 1 transport mean free path; roughly 1 mm of biological tissue. Our technique goes deeper into tissue, at nearly twice the depth,” details Seungwon Jeong.

While previous studies have looked at targets outside of the opaque environments, this study took it much further by placing the target in biological fluids. The target needs to reflect light more efficiently than its surroundings. For example, the scientists suggest that the technique could be used to visualize myelin, the lipid layer that wraps neurons and guarantees fast transmission of electrical signals, because it has a larger refractive index than the neurons it surrounds. The team hopes to apply this method to the nerve cells, bone marrow, and brain tissue of living animals and humans.

Letizia Diamante