New label-free deep-tissue imaging with wavelet correction algorithm extracts the fine neural network of the skull-intact mouse brain by focusing light and filtering out unwanted multiple scattered light waves – ScienceDaily

Researchers led by Associate Director CHOI Wonshik of the Center for Molecular Spectroscopy and Dynamics at the Institute of Basic Sciences, Professor KIM Moonseok of The Catholic University of Korea, and Professor CHOI Myunghwan of Seoul National University have developed a new type of holographic microscope. The new microscope is said to be able to “see through” the intact skull and is capable of high-resolution 3D imaging of the neural network in the brain of a living mouse without removing the skull.*

To probe 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 aberration1 appear when light hits the cells, making it difficult to get sharp images.

In complex structures such as living tissue, light undergoes multiple scattering, causing photons to randomly change direction multiple times as they travel through the tissue. Because of this process, much of the image information carried by the light is corrupted. However, even if this is a very small amount of reflected light, it is possible to observe features relatively deep in tissue by correcting the wavefront2 distortion of the light reflected from the target to be observed. However, the aforementioned multiple scattering effects interfere with this correction process. Therefore, to obtain high-resolution deep-tissue imaging, it is important to remove multiply scattered waves and increase the ratio of singly scattered waves.

Back in 2019, for the first time, IBS researchers developed a high-speed time-resolved holographic microscope3 which can 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 incision surgery. However, in the case of a mouse, which has a thicker skull than that of a fish, it was not possible to obtain an image of the brain from a neural network without removing or thinning the skull, due to strong light distortion and multiple scattering occurring , when light passes through the bone structure.

The research team was able to quantitatively analyze the interaction between light and matter, which allowed them to further improve their previous microscope. In this recent study, they report the successful development of an ultra-depth, three-dimensional, time-resolved holographic microscope that enables the observation of tissues at a greater depth than ever before.

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

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

Professor KIM Moonseok and Dr JO Yonghyeon, who developed the basis of the holographic microscope, said: “When we first observed the optical resonance of complex media, our work received a lot of attention from academia. From the basic principles to the practical application of observing a neural network under the mouse skull, we have discovered a new way for convergent brain neuroimaging technology by combining the efforts of talented people in physics, life and brain science.”

Associate Director CHOI Wonshik said, “For a long time, our Center has been developing super-depth bioimaging technology that applies physical principles. Our present discovery is expected to contribute significantly to the development of biomedical interdisciplinary research, including neuroscience and the industry of precision metrology.”

This research is published in the online edition of the journal Scientific progress (IF 14.136) on 28 July.

*For reference, a mouse skull has a similar thickness and opacity to a human fingernail.

Glossary of Terms:

1) Aberration is a phenomenon that occurs due to the change of the speed of light depending on the refractive index of the medium. This means that when the image is formed, all the light rays do not converge to a single point, causing the image to become blurry and distorted.

2) A wavefront refers to a plane that is formed by connecting all points of the same wave phase. For example, the wave front that is created when a stone is thrown into a lake is circular.

3) Time-Resolved Holographic Microscope: Holographic microscopy is a technology that detects the amplitude and phase of light using the light interference effect that occurs when two laser beams meet. In particular, a time-resolved holographic microscope can selectively acquire an optical signal at a certain depth by using a light source with a very short interference length of about 10 μm.

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