Holographic Microscope Achieves Through-Skull Brain Imaging

Researchers in South Korea have developed a new type of holographic microscope that can see through an intact skull. By focusing the light and filtering out undesired multiple scattered light waves, the technology was shown to be capable of high-resolution 3D imaging of the neural network within a living mouse brain.

To scrutinize the internal features of a living organism using light, it is necessary to deliver sufficient light energy to the sample and accurately measure the signal reflected from the target tissue. However, in living tissues multiple scattering effects and severe aberration tend to occur when light hits the cells, which makes it difficult to obtain sharp images. When there is a very small amount of reflected light, it is possible to observe the features located relatively deep within the tissues by correcting the wavefront distortion of the light that was reflected from the target. However, multiple scattering effects also interfere with this correction process. Therefore, to obtain a high-resolution deep-tissue image, it is important to remove the multiple-scattered waves and increase the ratio of the single-scattered waves.

In 2019, the team from Institute of Basic Science (IBS) were the first to develop high-speed time-resolved holographic microscope that can eliminate multiple scattering and simultaneously measure the amplitude and phase of light. At the time, they used this microscope to observe the neural network of live fish without incisional surgery. However, in the case of a mouse, which has a thicker skull than that of a fish, it was not possible to obtain a neural network image of the brain without removing or thinning the skull, due to severe light distortion and multiple scattering occurring when the light travels through the bone structure.

In their most recent work—led by Associate Director Choi Wonshik of the Center for Molecular Spectroscopy and Dynamics within IBS, Professor Kim Moonseok of The Catholic University of Korea, and Professor Choi Myunghwan of Seoul National University—the team managed to quantitatively analyze the interaction between light and matter, which allowed them to further improve their previous microscope. They report  in Science Advances the successful development of a super-depth, three-dimensional time-resolved holographic microscope that allows for the observation of tissues to a greater depth than ever before.

Specifically, the researchers devised a method to preferentially select single-scattered waves by taking advantage of the fact that they have similar reflection waveforms even when light is input from various angles. This is done by a complex algorithm and a numerical operation that analyzes the eigenmode of a medium (a unique wave that delivers light energy into a medium), which allows the finding of a resonance mode that maximizes constructive interference (interference that occurs when waves of the same phase overlap) between wavefronts of light. This enabled the new microscope to focus more than 80 times of light energy on the neural fibers than before, while selectively removing unnecessary signals. This allowed the ratio of single-scattered waves versus multiple-scattered waves to be increased by several orders of magnitude.

The research team went on the demonstration of this new technology by observing the mouse brain. The microscope was able to correct the wavefront distortion even at a depth that was previously impossible using existing technology. The new microscope succeeded in obtaining a high-resolution image of the mouse brain's neural network under the skull. This was all achieved in the visible wavelength without removing the mouse skull and without requiring a fluorescent label.

“When we first observed the optical resonance of complex media, our work received great attention from academia,” according to Moonseok and Yonghyeon. “From basic principles to practical application of observing the neural network beneath the mouse skull, we have opened a new way for brain neuroimaging convergent technology by combining the efforts of talented people in physics, life, and brain science.”

Wonshik says the breakthrough “will greatly contribute to the development of biomedical interdisciplinary research including neuroscience and the industry of precision metrology.”