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Advanced CLARITY for rapid and high-resolution imaging of intact tissues


CLARITY is a method for chemical transformation of intact biological tissues into a hydrogel-tissue hybrid, which becomes amenable to interrogation with light and macromolecular labels while retaining fine structure and native biological molecules. This emerging accessibility of information from large intact samples has created both new opportunities and new challenges. Here we describe protocols spanning multiple dimensions of the CLARITY workflow, ranging from simple, reliable and efficient lipid removal without electrophoretic instrumentation (passive CLARITY) to optimized objectives and integration with light-sheet optics (CLARITY-optimized light-sheet microscopy (COLM)) for accelerating data collection from clarified samples by several orders of magnitude while maintaining or increasing quality and resolution. The entire protocol takes from 7–28 d to complete for an adult mouse brain, including hydrogel embedding, full lipid removal, whole-brain antibody staining (which, if needed, accounts for 7–10 of the days), and whole-brain high-resolution imaging; timing within this window depends on the choice of lipid removal options, on the size of the tissue, and on the number and type of immunostaining rounds performed. This protocol has been successfully applied to the study of adult mouse, adult zebrafish and adult human brains, and it may find many other applications in the structural and molecular analysis of large assembled biological systems.

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Figure 1: CLARITY pipeline overview.
Figure 2: ETC.
Figure 3: Imaging clarified samples.
Figure 4: COLM for large intact samples.
Figure 5: Ultrafast imaging of whole mouse brain using COLM.
Figure 6: Fast high-resolution imaging of clarified brain using COLM.
Figure 7: Optimized confocal microscopy of clarified samples.
Figure 8: Molecular interrogation of clarified tissue.


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We thank the entire Deisseroth laboratory for helpful discussions and Anna Lei for technical assistance. K.D. is supported by the Defense Advance Research Projects Agency (DARPA) Neuro-FAST program, the National Institute of Mental Health (NIMH), the National Science Foundation (NSF), the National Institute on Drug Abuse (NIDA), the Simons Foundation and the Wiegers Family Fund. All CLARITY tools and methods described are distributed and supported freely (, and discussed in an open forum (

Author information

Authors and Affiliations



R.T. and K.D. wrote the paper with input from L.Y. on the content. R.T., L.Y. and B.H. prepared the clarified samples, and R.T. developed the imaging framework. K.D. supervised the project.

Corresponding author

Correspondence to Karl Deisseroth.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Sample transparency at key stages of the CLARITY protocol.

Pictures of representative brain samples are shown during the key stages of clarification: (a) hydrogel-embedded mouse brain, (b) after lipid removal, (c) after washing of the residual SDS micelle with PBST, and (d) after 2 hours of refractive index homogenization with FocusClear at 37°C with gentle shaking.

Supplementary Figure 2 Summary of the control electronics framework and COLM parts.

FPGA logic is used to control and synchronize various parts of the microscope.

Supplementary information

Supplementary Figure 1

Sample transparency at key stages of the CLARITY protocol. (PDF 513 kb)

Supplementary Figure 2

Summary of the control electronics framework and COLM parts. (PDF 422 kb)

Volume rendering of whole mouse brain volume acquired from an intact clarified Thy1-eYFP mouse brain using 10× magnification objective in COLM.

The dataset was down-sampled 4 fold in each lateral pixel dimension to make the volume rendering feasible with current computing power. The entire image volume was acquired in 4 hours. (MOV 54043 kb)

Rendering of 3.15 mm × 3.15 mm × 5.3 mm volume acquired from an intact clarified Thy1-eYFP mouse brain using the 25× magnification objective in COLM.

The dataset was down-sampled 2 fold in each lateral pixel dimension. The entire image volume was acquired in 1.5 hours. (MOV 39475 kb)

Rendering of 1.837 mm × 0.959 × 5 mm volume acquired from an intact clarified Thy1-eYFP mouse brain using the 25× magnification objective in a confocal microscope. (MOV 27073 kb)

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Tomer, R., Ye, L., Hsueh, B. et al. Advanced CLARITY for rapid and high-resolution imaging of intact tissues. Nat Protoc 9, 1682–1697 (2014).

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