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Three-dimensional imaging of solvent-cleared organs using 3DISCO

Abstract

The examination of tissue histology by light microscopy is a fundamental tool for investigating the structure and function of organs under normal and disease states. Many current techniques for tissue sectioning, imaging and analysis are time-consuming, and they present major limitations for 3D tissue reconstruction. The introduction of methods to achieve the optical clearing and subsequent light-sheet laser scanning of entire transparent organs without sectioning represents a major advance in the field. We recently developed a highly reproducible and versatile clearing procedure called 3D imaging of solvent-cleared organs, or 3DISCO, which is applicable to diverse tissues including brain, spinal cord, immune organs and tumors. Here we describe a detailed protocol for performing 3DISCO and present its application to various microscopy techniques, including example results from various mouse tissues. The tissue clearing takes as little as 3 h, and imaging can be completed in 45 min. 3DISCO is a powerful technique that offers 3D histological views of tissues in a fraction of the time and labor required to complete standard histology studies.

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Figure 1: Typical results obtained from 3DISCO of neurons in the brain and hippocampus.
Figure 2: Clearing reagents and steps.
Figure 3: Ultramicroscopy setup.
Figure 4: 3DISCO with wide-field fluorescence, confocal and two-photon microscopy.
Figure 5: Typical results obtained from 3DISCO of vasculature in the brain and spinal cord.
Figure 6: Typical results obtained from 3DISCO of lung tumors and mammary glands.
Figure 7: Typical results obtained from 3DISCO of immune organs.

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Acknowledgements

We thank M. Chang and F. Yeh for critically reading the manuscript, and C. Chalouni, H. Ngu and L. Komuves for assistance with large-scale imaging microscopy and macros. We thank J. Sanes (Washington University in St. Louis) for the Thy-1 GFP (GFP-M) line, T. Jacks (Massachusetts Institute of Technology) for KrasLSLG12D mice, H. Ploegh (Massachusetts Institute of Technology) for MHCII-GFP mice, M. Nussenzweig (Rockefeller University) for CD11c YFP-Venus mice, and D.R. Littman (New York University) for CX3CR1-GFP mice. We thank C. Murriel (Genentech) for providing the lung tissues and C. Sakanaka (Genentech) for providing mammary gland tissues. The work is supported by Genentech and the Hertie foundation. N. Jährling was supported by the Theodor Körner Fonds.

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Authors

Contributions

A.E. initiated the current project, designed and performed all the experiments, analyzed the data, and made all the figures and videos. A.E. and M.S. wrote the paper. C.D.H. prepared the immune organs and J.G.E. provided transgenic mice. K.B. and N.J. developed and performed various clearing protocols. A.E., K.B., N.J., C.P.M., F.H., F.B., M.S. and H.-U.D. contributed to the development of the clearing protocol at various stages. H.-U.D. led the development of the ultramicroscopy and clearing technology. All authors edited the paper.

Corresponding author

Correspondence to Ali Ertürk.

Ethics declarations

Competing interests

A.E., C.D.H., J.G.E. and M.S. are employees of Genentech, a member of the Roche Group.

Supplementary information

Supplementary video 1

A cleared spinal cord tissue of a GFP-M mouse imaged with 2-photon microscopy. In this high resolution scans, the individual axons can be readily traced. (MOV 15907 kb)

Supplementary video 2

The simulation of a scan through entire cleared brain from a GFP-M mouse in two different orientations: horizontal and sagittal. The bottom two windows show the horizontal scan in higher magnifications (left showing the hippocampus and right showing the cerebellum). (MOV 9533 kb)

Supplementary video 3

3D reconstruction and animation of the hippocampus from a GFP-M mouse. (MOV 9680 kb)

Supplementary video 4

3D reconstruction and animation of the spinal cord vasculature from a wild type mouse traced with lectin-FITC labeling. (MOV 18079 kb)

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Ertürk, A., Becker, K., Jährling, N. et al. Three-dimensional imaging of solvent-cleared organs using 3DISCO. Nat Protoc 7, 1983–1995 (2012). https://doi.org/10.1038/nprot.2012.119

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