Research Findings

2025.08.26

SeeThrough: a rationally designed skull clearing technique for in vivo brain imaging

▶ Nat Commun. 2025 Aug 26;16(1):7584. doi:10.1038/s41467-025-62836-1.

Liu X#,1,2, Uchigashima M#,1,3, Oomoto I4, Saito Y4, Uchida H2, Oginezawa S1,5, Masuda K6, Satoh D1, Abe M7, Sakimura K7, Shimizu Y6, Murayama M8, Tainaka K9, Mikuni T10.

  1. Department of Cellular Neuropathology, Brain Research Institute, Niigata University
  2. Department of System Pathology for Neurological Disorders, Brain Research Institute, Niigata University
  3. International Research Center for Neurointelligence (WPI-IRCN), The University of Tokyo, Tokyo
  4. Center for Brain Science, RIKEN
  5. Department of Neurology, Brain Research Institute, Niigata University
  6. Laboratory for Cell-Free Protein Synthesis, RIKEN Center for Biosystems Dynamics Research
  7. Department of Animal Model Development, Brain Research Institute, Niigata University
  8. Center for Brain Science, RIKEN. masanori.murayama@riken.jp.
  9. Department of System Pathology for Neurological Disorders, Brain Research Institute, Niigata University. kztainaka@bri.niigata-u.ac.jp.
  10. Department of Cellular Neuropathology, Brain Research Institute, Niigata University. tmikuni@bri.niigata-u.ac.jp.

#Contributed equally.

Abstract

Light scattering in the skull limits optical access to the brain. Here we present SeeThrough, a skull-clearing technique that enables simple, high-resolution, and minimally-invasive brain imaging without skull removal. Through systematic screening of over 1600 chemicals, we rationally developed a refractive index-matching solution that combines water- and organic solvent-based components, achieving both high clearing efficiency and biocompatibility. The reagents exhibit minimal brain penetration, maintain tissue integrity, and avoid inflammatory responses. Notably, SeeThrough provides imaging sensitivity and contrast comparable to open-skull window imaging, while permitting minimally-invasive monitoring of brain border macrophages as well as blood and cerebrospinal fluid dynamics. Combined with two-photon imaging, SeeThrough enables spatially and temporally scalable imaging applications in the mouse brain, including ~400 µm deep imaging, one-month longitudinal imaging, and mesoscale, cellular-resolution monitoring of brain activity for network-level analysis. Thus, SeeThrough offers a broadly accessible platform for high-throughput, physiology-preserving imaging of the brain parenchyma and brain-skull interface.

*Reprinted under a CC BY NC ND 4.0 license.

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