A rapid optical clearing protocol using 2,2'-thiodiethanol for microscopic observation of fixed mouse brain

PLoS One. 2015 Jan 29;10(1):e0116280. doi: 10.1371/journal.pone.0116280. eCollection 2015.


Elucidation of neural circuit functions requires visualization of the fine structure of neurons in the inner regions of thick brain specimens. However, the tissue penetration depth of laser scanning microscopy is limited by light scattering and/or absorption by the tissue. Recently, several optical clearing reagents have been proposed for visualization in fixed specimens. However, they require complicated protocols or long treatment times. Here we report the effects of 2,2'-thiodiethanol (TDE) solutions as an optical clearing reagent for fixed mouse brains expressing a yellow fluorescent protein. Immersion of fixed brains in TDE solutions rapidly (within 30 min in the case of 400-µm-thick fixed brain slices) increased their transparency and enhanced the penetration depth in both confocal and two-photon microscopy. In addition, we succeeded in visualizing dendritic spines along single dendrites at deep positions in fixed thick brain slices. These results suggest that our proposed protocol using TDE solution is a rapid and useful method for optical clearing of fixed specimens expressing fluorescent proteins.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Brain / ultrastructure*
  • Dendrites / ultrastructure*
  • Dendritic Spines / ultrastructure
  • Mice
  • Microscopy, Confocal
  • Neurons / ultrastructure*
  • Sulfhydryl Compounds*


  • Sulfhydryl Compounds
  • 2,2'-thiodiethanol

Grants and funding

This work was supported by Grant-in-Aid for JSPS Fellows (No. 26-1533 for YA) (http://www.jsps.go.jp/english/), by JSPS KAKENHI grants of the Ministry of Education, Culture, Sports, Science and Technology, Japan (No. 26242082 and 22113005 for TN) (MEXT; http://www.mext.go.jp/english/), by the Nano-Macro Materials, Devices and System Research Alliance (MEXT; http://www.mext.go.jp/english/) and by the Network Joint Research Center for Materials and Devices (MEXT; http://www.mext.go.jp/english/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.