Professor Takayasu Mikuni of the Department of Cellular Neuropathology and Professor Kazuki Tainaka of the Department of System Pathology for Neurological Disorders at Niigata University's Brain Research Institute, together with Team Director Masanori Murayama of RIKEN's Center for Brain Science and their colleagues, announced on August 26 that they have jointly developed the "SeeThrough method," which can make the skulls of living animals transparent. They developed a new skull clearing reagent and confirmed that simply applying it can reversibly make the skulls of 2-3 month-old living mice transparent with over 95% transmittance. This enables minimally invasive live imaging of the brains of living animals without skull thinning, and an increased understanding of brain information processing is expected. The results were published in Nature Communications on August 26.
Two-photon microscopy allows observation of the dynamics of tissues and organs inside living experimental animals. As it enables observation of changes in nerve cells associated with behavior and disease, it is widely used in brain science research.
Previously, this technique required advanced procedures to observe mouse brains under opaque skulls, either by thinning the skull (thinned skull method) or removing part of the skull and inserting glass (open-skull method). However, the thinned skull method results in bone regeneration within a few days, while the open-skull method has high invasiveness and cannot avoid damage to brain parenchyma. Furthermore, neither technique could create large observation windows covering the entire cerebral cortex. For large-scale brain network analysis, less invasive methods capable of observing wider areas were needed.
Although clearing reagents that achieve transparency by changing refractive index have been reported, their clearing efficiency was low, at around 50%. Therefore, the research group aimed to develop skull clearing reagents that combine both biocompatibility and transparency.
For biological tissue clearing, it is necessary to match the refractive index of the reagent to that of the tissue. There are two types of clearing reagent: organic solvent-based and water-soluble. Organic solvent-based reagents have high refractive indices but low biocompatibility, while water-soluble reagents have high biocompatibility but low refractive indices, presenting challenges for transparency.
To achieve both biocompatibility and transparency, the group investigated combinations of organic solvent-based and water-soluble reagents. They theoretically and experimentally screened over 1,600 compounds. As a result, they were able to find a candidate reagent that is mixable, biocompatible, and matches the high refractive index of the skull (1.56).
When they verified whether the reagent could actually clear the skull using isolated skull sections, they confirmed that it could achieve transparency with over 95% transmittance. It achieved transparency comparable to organic solvent-based reagents.
To verify it with actual living mice, the group made incisions only in the skin of the mouse crown and applied the reagent, resulting in skull transparency within 60 minutes. Blood vessels and dendritic processes of nerve cells could be clearly observed to the same extent as with the open-skull method.
The reagent was reusable and enabled them to capture temporal changes in dendritic spines. To confirm its invasiveness, they compared inflammatory cell counts 24 hours after each treatment with the SeeThrough method and open-skull method. The SeeThrough method showed no change in cell count, while the open-skull method showed significant increase. When they examined astrocyte activation caused by inflammation, there was no difference between untreated mice and the SeeThrough method, while the open-skull method showed significant activation.
When they performed deep cerebral cortex imaging of living mice using this reagent, they succeeded in deep brain imaging reaching pyramidal cells in layer 5 at 400 µm from the brain surface. Furthermore, they simultaneously captured the activity of 3,000 nerve cells existing in wide areas of the cerebral cortex and confirmed that neural network analysis was possible. They also succeeded in confirming the existence of long-range networks and hub neurons.
Mikuni commented: "We have developed skull clearing technology: the 'SeeThrough method.' This has created foundational technology for simple, minimally invasive, high-precision live brain imaging. We hope this will become widespread in the future. Also, by observing the living brain in physiological conditions without physical damage, we expect to capture brain phenomena more accurately than ever before."
Journal Information
Publication: Nature Communications
Title: SeeThrough: a rationally designed skull clearing technique for in vivo brain imaging
DOI: 10.1038/s41467-025-62836-1
This article has been translated by JST with permission from The Science News Ltd. (https://sci-news.co.jp/). Unauthorized reproduction of the article and photographs is prohibited.