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In vivo optical clearing of the mouse brain

Holy, T. E.; Kume, M.; Kang, N.; Akrouh, A.; Kim, D. W.; Dearborn, J. T.; Wozniak, D. F.; Kerschensteiner, D.

2026-08-23 neuroscience
10.64898/2026.08.18.745591 bioRxiv
Show abstract

Light microscopy is one of the most powerful tools for understanding living systems, but the opacity of tissue prevents visualization of all but superficial layers. Several methods to clarify tissue have been developed, but most require fixed specimens. To address the challenge of improving resolution in functioning neuronal circuits, we developed a biocompatible clearing agent, iodixanol-ACSF, which is capable of increasing the transparency of living neuronal tissue. Brain-cleared mice were motile and unimpaired on a variety of behavioral tasks, and extracellular recordings showed that many cellular and circuit phenomena were well-preserved. In live iodixanol-ACSF cleared mouse brain tissue, both transmission and cellular-resolution fluorescence microscopy indicate improvements of 150-200% in penetration depth with one-third to one-half the laser intensity when compared to untreated tissue. Our results show that iodixanol-ACSF clearing will enable deeper imaging and extend our understanding of neuronal circuit function.

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