A research group led by Professor Takeshi Imai and Assistant Professor Shigenori Inagaki from the Faculty of Medical Sciences at Kyushu University, together with Kagoshima University and the University of Yamanashi, has become the first in the world to render a living mouse brain transparent and repeatedly observe it while maintaining normal biological functions. Imai commented: "When we first made postmortem specimen mouse brains transparent more than a decade ago, people often asked when we would be able to do it with a living mouse. We have finally managed it. Since we can now observe the brain without affecting its functions, I hope researchers will make use of this in a wide variety of studies." Their findings were published in Nature Methods.
(A, B) Clearing of live brain tissue. Live brain tissue after immersion in standard medium (A) and SeeDB-Live (B) for approximately 1 hour, respectively. (C) Fluorescent calcium measurement in live brain tissue.
Provided by Assistant Professor Shigenori Inagaki, Professor Takeshi Imai of Kyushu University
The main reason living tissue appears opaque is that light refracts and scatters as it passes through and around cells, preventing it from traveling in a straight line. However, conventional tissue-clearing reagents have high concentrations that cause cells to lose water through osmotic pressure, killing them.
The research group tested various molecules to find the optimal tissue-clearing reagent. They found that adding albumin, a type of protein, to the extracellular fluid and suppressing light refraction and scattering allowed them to render living tissue transparent in a non-invasive manner. Albumin barely alters the ionic composition of the extracellular fluid and has no cytotoxicity. This made it possible to use fluorescence microscopy to observe normal cellular functions within transparentized living tissue for the first time ever.
Inagaki recalled: "I started working on this around 2021, and within a few months, I concluded that spherical polymers seemed to be the way to go. However, the candidate molecules were adversely affecting cell function, and I was stuck for about a year. When I was working alone in the lab late at night and there was no one around, I tried a highly pure, expensive albumin reagent—and it worked."
Following experiments with live cells, the group introduced their newly developed clearing reagent, SeeDB-Live, into the brains of living mice and successfully imaged them down to layer 5 of the cerebral cortex using fluorescence microscopy. The mice remained normal even after repeated experiments spanning more than four months. The researchers plan to improve the method of penetrating the reagent further so that even deeper layers can be visualized in the future.
(A) Clearing of a living mouse brain. In an anesthetized mouse, SeeDB-Live was continuously applied to the brain surface for 1 hour to allow it to penetrate. (B) Three-dimensional fluorescent image of neurons in layer V of the cerebral cortex. (C-E) Dendrites and dendritic spines of neurons in layer V of the cerebral cortex.
Provided by Assistant Professor Shigenori Inagaki, Professor Takeshi Imai of Kyushu University
Journal Information
Publication: Nature Methods
Title: Isotonic and minimally invasive optical clearing media for live cell imaging ex vivo and in vivo
DOI: 10.1038/s41592-026-03023-y
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.