Holographic microscope can see through the skull, image the brain

A new type of holographic microscope, developed by South Korean researchers, is reportedly capable of seeing through the intact skull and enabling high-resolution 3D imaging of the neural network within a living mouse brain, without removing the skull. The breakthrough has been reported in the journal Science Advances.

In order to scrutinise the internal features of a living organism using light, it is necessary to a) deliver sufficient light energy to the sample and b) 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.

In complex structures such as living tissue, light undergoes multiple scattering, which causes the photons to randomly change their direction several times as they travel through the tissue. Because of this process, much of the image information carried by the light becomes ruined. However, even if it 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 to be observed. However, the abovementioned multiple scattering effects interfere with this correction process. Therefore, in order 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.

Back in 2019, researchers at Korea’s Institute for Basic Science (IBS) developed a high-speed time-resolved holographic microscope that could eliminate multiple scattering and simultaneously measure the amplitude and phase of light. 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.

The research team managed to quantitatively analyse the interaction between light and matter, which allowed them to further improve their previous microscope. In this recent study, they reported the successful development of a super-depth, three-dimensional, time-resolved holographic microscope that is said to allow 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 analyses the eigenmode of a medium (a unique wave that delivers light energy into a medium), which allows the finding of a resonance mode that maximises 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 fibres 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.

Demonstrating this new technology by observing a mouse brain, the microscope was able to correct the wavefront distortion even at a depth that was previously impossible using existing technology. The microscope succeeded in obtaining a high-resolution image of the brain’s neural network under the skull — all 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,” said Professor Moonseok Kim from The Catholic University of Korea and Dr Yonghyeon Jo from the IBS Center for Molecular Spectroscopy and Dynamics (CMSD), who developed the foundation of the holographic microscope. “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.”

CMSD Associate Director Wonshik Choi added, “For a long time, our centre has developed super-depth bioimaging technology that applies physical principles. It is expected that our present finding will greatly contribute to the development of biomedical interdisciplinary research including neuroscience and the industry of precision metrology.”

Image caption: A neural network in the brain of a living mouse was observed without removing the skull.

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