Isotonic and minimally invasive optical clearing media for live cell imaging ex vivo and in vivo
Tissue clearing has been widely used for fluorescence imaging of fixed tissues, but its application to live tissues has been limited by toxicity. Here we develop minimally invasive optical clearing media for fluorescence imaging of live mammalian tissues. Light scattering is minimized by adding spherical polymers with low osmolarity to the extracellular medium. A clearing medium containing bovine serum albumin (SeeDB-Live) is compatible with live cells, enabling structural and functional imaging of live tissues, such as spheroids, organoids, acute brain slices and the mouse brains in vivo. SeeDB-Live minimally affects neuronal electrophysiological properties and sensory responses in vivo, and facilitates fluorescence imaging of deep cortical layers in live animals without detectable toxicity to neurons or behavior. We further demonstrate its utility to epifluorescence voltage imaging in acute brain slices and in vivo preparations. Thus, SeeDB-Live expands both the depth and modality range of fluorescence imaging in live mammalian tissues.
Authors
- Yuki Kambe
- Nao Nakagawa-Tamagawa (ORCID: https://orcid.org/0000-0002-8888-9240)
- Tatsushi Yokoyama (ORCID: https://orcid.org/0000-0001-7159-9039)
- Shigenori Inagaki (ORCID: https://orcid.org/0000-0003-1413-1207)
- Yoshiaki Tagawa (ORCID: https://orcid.org/0000-0002-3300-090X)
- Masayuki Sakamoto (ORCID: https://orcid.org/0000-0001-6458-113X)
- K. Kitamura (ORCID: https://orcid.org/0000-0002-8956-4122)
- Satoshi Fujimoto (ORCID: https://orcid.org/0000-0002-7618-5502)
- Satoshi Manita (ORCID: https://orcid.org/0000-0002-4678-0853)
- Satoru Okuda (ORCID: https://orcid.org/0000-0003-3792-8067)
- Katsuhiko Hayashi (ORCID: https://orcid.org/0000-0001-7359-5939)
- Tatsuo K. Sato (ORCID: https://orcid.org/0000-0002-1279-5125)
- Yuki Naitou (ORCID: https://orcid.org/0000-0002-8470-5539)
- Takeshi Imai (ORCID: https://orcid.org/0000-0002-8449-0080)
- Aki Teranishi (ORCID: https://orcid.org/0009-0005-7209-010X)
- Rei Yagasaki
- Takahiro Noda
- Misato Mori
- Hikari Takeshima
- Koki Ishikawa
- Nathan Zechen Huynh
Institutions
- Kanazawa University (JP)
- Kagoshima University (JP)
- Kyushu University (JP)
- Kyoto University (JP)
- Japan Science and Technology Agency (JP)
- Kyoto College of Medical Science (JP)
- Life Science Institute (JP)
- University of Yamanashi Hospital (JP)
- University of Yamanashi (JP)
- The University of Osaka (JP)
Publication Details
- Journal
- Nature Methods
- Published
- 2026-03-12
- DOI
- https://doi.org/10.1038/s41592-026-03023-y
- Primary Topic
- Advanced Fluorescence Microscopy Techniques
- Type
- article
- Field-Weighted Citation Impact
- 0.00