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Method Article
Deep Imaging of Cleared Brain by Confocal Laser-Scanning Microscopy
Atsushi Miyawaki
Laboratory for Cell Function Dynamics, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-city, Saitama, 351-0198, Japan
Corresponding Author
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The Sca_l_eS technique for clearance of biological tissue enables us to perform high-resolution three-dimensional (3D) reconstructions of entire structures in fixed, unsectioned brain samples where specific cell types are labeled with fluorescent proteins genetically or with fluorophore-conjugated antibodies. While Sca_l_eA2 and Sca_l_eU2, the previously reported urea-based clearing solutions have refractive indices (RIs) of 1.38 and 1.40, respectively, Sca_l_eS solutions containing sorbitol in addition to urea have higher RIs around 1.47. Importantly, Sca_l_eS solutions preserve fluorescence signals and tissue fine structures, allowing quantitative three-dimensional reconstructions. Here we provide a basic protocol describing the procedures for solution preparation, sample clarification and immobilization for observation using confocal laser-scanning microscopy. We emphasize the importance of RI matching by showing results of comparative experiments that deep imaged YFP-expressing neurons within cleared brains by Sca_l_eS, Sca_l_eA2, or Sca_l_eU2 and utilized objective lenses with long working distances, sufficiently high numerical apertures, but different RIs.
Minor edits requested by the author were made to the formatting of the protocol on 5th April 2016. The original version of the protocol is available as an attachment. Katharine Barnes, Managing Editor, Nature Protocols, 5/4/16
Keywords
Tissue clearing, Urea, Refractive index, Laser-scanning microscopy
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This is a list of supplementary files associated with this preprint. Click to download.
- supplement0.xlsx
*Table 1* *Preparation of reagents* A new version of this table was uploaded on 5th April 2016 as Revised Table 1.
- supplement0.xlsx
*Revised Table 1* *Preparation of reagents version 2* This version of Table 1 was uploaded on 5th April 2016. A new version of this table was uploaded on 28th November 2016.
- supplement0.xlsx
*Revised Table 1 (2)* *Preparation of reagents version 3* This version of Table 1 was uploaded on 28th November 2016.
- supplement0.pdf
*Version 1 of protocol* *Version 1 of protocol* This is the version of the protocol that was live initially. Minor edits were made to the protocol and pushed live on 5th April 2016.
- supplement0.pdf
*Version 2 of protocol* *Version 2 of protocol* This is the version of the protocol that was pushed live on 5th April 2016.
- supplement0.xlsx
*Table 2* *Guide for clearing of mouse brain tissues.*
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Associated Publications
ScaleS: an optical clearing palette for biological imaging
by Hiroshi Hama, Hiroyuki Hioki, Kana Namiki, +8
Nature Neuroscience (15 March, 2016)
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This is a preprint. It has not completed peer review.
Method Article
Deep Imaging of Cleared Brain by Confocal Laser-Scanning Microscopy
Atsushi Miyawaki
Laboratory for Cell Function Dynamics, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-city, Saitama, 351-0198, Japan
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 24 Mar, 2016
No community comments so far
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a CC BY-NC 3.0 License. Read Full License
The Sca_l_eS technique for clearance of biological tissue enables us to perform high-resolution three-dimensional (3D) reconstructions of entire structures in fixed, unsectioned brain samples where specific cell types are labeled with fluorescent proteins genetically or with fluorophore-conjugated antibodies. While Sca_l_eA2 and Sca_l_eU2, the previously reported urea-based clearing solutions have refractive indices (RIs) of 1.38 and 1.40, respectively, Sca_l_eS solutions containing sorbitol in addition to urea have higher RIs around 1.47. Importantly, Sca_l_eS solutions preserve fluorescence signals and tissue fine structures, allowing quantitative three-dimensional reconstructions. Here we provide a basic protocol describing the procedures for solution preparation, sample clarification and immobilization for observation using confocal laser-scanning microscopy. We emphasize the importance of RI matching by showing results of comparative experiments that deep imaged YFP-expressing neurons within cleared brains by Sca_l_eS, Sca_l_eA2, or Sca_l_eU2 and utilized objective lenses with long working distances, sufficiently high numerical apertures, but different RIs.
Minor edits requested by the author were made to the formatting of the protocol on 5th April 2016. The original version of the protocol is available as an attachment. Katharine Barnes, Managing Editor, Nature Protocols, 5/4/16
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Associated Publications
ScaleS: an optical clearing palette for biological imaging
by Hiroshi Hama, Hiroyuki Hioki, Kana Namiki, +8
Nature Neuroscience (15 March, 2016)