Hologram Microscope Developed to Penetrate Neural Network in Brain
80 times more light collected from inside the skull of a live mouse allowing observation of the neural network without use of fluorescent markers
Technology expected to be applied to various convergence studies in medical life sciences and industries requiring precise measurement.
▲ (From left top, clockwise) Wonshik Choi, Vice-president of the Center for Molecular Spectroscopy and Dynamics at Institute for Basic Science (IBS) (co-corresponding author); Moonseok Kim, assistant professor in the Department of Medical Life Sciences of College of Medicine at Catholic University of Korea (co-corresponding author); Ye-Ryoung Lee, researcher in the Center for Molecular Spectroscopy and Dynamics at IBS (co-first author); and Yonghyeon Jo, researcher in the Center for Molecular Spectroscopy and Dynamics at IBS (co-first author).
The joint research group, consisting of Wonshik Choi (Professor in the Department of Physics at KU), Vice-president of the Center for Molecular Spectroscopy and Dynamics at IBS (President: Professor Min-haeng Cho in the Department of Chemistry at KU), Professor Moonseok Kim of Catholic University of Korea, and Professor Myung-hwan Choi of Seoul National University developed a hologram microscope capable of three-dimensional observation of the brain neural network of a living mouse at high resolution without removal of mouse skull.
Observation of deep parts of the human body using light requires the transfer of a sufficiently large amount of light energy and accurate measurement of the reflected signals. However, this type of observation is difficult in living tissues because of the multiple scattering caused by the collision of light with the varied cells and the aberration leading to image blur.
* Aberration: Blurring or obscuring of an image that occurs when light is not focused on a single point because of the different arrival times of light caused by their different refractivity.
In complicated structures, such as living tissues, light undergoes multiple scattering events during which the direction of light propagation is randomly changed many times. This process destroys the imaging information of the light. However, if the light that is reflected only one time from the object being observed (single-scattered waves) can be selected for, even if only a very small amount is collected, this may be used to correct wavefront distortion and observe deep structures. However, multiple-scattered waves disturb this process. Therefore, when acquiring deep-tissue images, it is important to remove the disturbing multiple-scattered waves and thereby increase the proportion of single-scattered waves.
* Wavefront: A surface formed by connecting all points having the same phase in a wave. For example, the wavefront of the water wave formed by throwing a stone onto a calm lake is circular.
In 2019 the research group of IBS developed the world’s first time-resolved hologram microscope, which is capable of removing multiple scattering and simultaneously measuring light intensity and phase, and were able to observe the neural network of a living fish without an excisional operation. However, in the case of mice, which have thicker skulls than fish, the severe distortion and multiple scattering of light in the skull made images of the brain neural network unobtainable without first removing or clipping off skull.
* Hologram microscope: A technology for investigating the amplitude and phase of light based on the interference of two laser rays. A time-resolved hologram microscope, which employs a light source featuring an extremely short interference length (about 10 μm), is capable of selectively acquiring optical signals from a specific depth.
The research group developed a deeper 3D time-resolved hologram microscope able to observe deeper structures by quantizing the interactions between light and materials. The group developed a method of selecting only single-scattered waves based on their similar reflective waveforms despite their different incident angles. In this method, a numerical simulation for analyzing the natural mode of the medium (the material transmitting the waves) is carried out to determine the resonance state maximizing constructive interference (the interference caused by the overlapping of waves of the same phase) between the light and the wavefront. The research group were able to collect 80 times more light from the brain neural network through this approach than has been achieved through previously used techniques, and could selectively remove unnecessary signals, increasing the proportion of single-scattered waves by dozens of times.
The research group corrected light wavefront distortion at a depth impossible using conventional technologies, and successfully obtained high-resolution images of the brain neural network under the skull at visible wavelengths without removing mouse skull or applying fluorescent marker.
Professor Kim and Professor Cho, who developed the foundation of the hologram microscope, commented, “We received much attention from the academic field when we first observed the optical resonance state of complicated materials. We have been able to work together with researchers in physics, life sciences and brain sciences to investigate basic principles and observe the neural network inside the mouse skull, opening a new way toward convergence technologies in the field of brain neural imaging.”
Wonshik Choi, the Vice-president of the Center, said, “The deep bioimaging technology based on physical principles that we have long investigated will make great contributions to the development of optical microscope imaging technology.” He added, “We are looking forward to seeing the ripple effects in various convergence studies in life sciences, including brain neural science and industrial fields requiring precise measurement.”
The research results were published in Science Advances (IF 14.136), an internationally renowned journal, on July 28 in the online edition.
- Title : Through-skull brain imaging in vivo at visible wavelengths via dimensionality reduction adaptive-optical microscopy
- Authors : Yonghyeon Jo, Ye-Ryoung Lee, Jin Hee Hong, Dong-Young Kim, Junhwan Kwon, Myunghwan Choi, Moonseok Kim and Wonshik Choi
[ Figure Explanation ]
[Figure 1] A deep 3D hologram microscope
The deep 3D hologram microscope developed by the research group of the Center for Molecular Spectroscopy and Dynamics at IBS. The target optical signal ratio was increased and the image acquisition rate and depth were also increased to allow observation of the neural network of even living organisms.
[Figure 2] Characteristics of reflective signals depending on the incident angles
When an object has a small size or a linear structure, the waveforms of the reflected signals of the single-scattered waves, measured at different incident angles, remain similar to each other (A). Conversely, the waveforms of the reflected signals of the multiple-scattered waves lack any similarity (B). These characteristics of the different wavefronts can be used to separate the single-scattered components from the multiple-scattered components.
[Figure 3] Brain neural network of a living mouse observed without removing the skull.
The brain neural network images were successfully obtained using a light source at visible wavelengths by simply removing the skin of a living mouse in the presence of the skull (A). With the conventional technology, brain neural network images were unobtainable because aberrations could not be corrected for due to the many multiple-scattered waves generated in the skull (B). However, the research group applied the newly developed technology to selectively remove only the multiple-scattered components to determine the aberration (D), and then corrected the aberration to obtain images of the brain neural network (C).