Abstract

Recent tissue-clearing approaches have become important alternatives to standard histology approaches. However, light scattering in thick tissues and the size restrictions on samples that can be imaged with standard light-sheet microscopy pose limitations for analyzing large samples such as an entire rodent body. We developed 'ultimate DISCO' (uDISCO) clearing to overcome these limitations in volumetric imaging. uDISCO preserves fluorescent proteins over months and renders intact organs and rodent bodies transparent while reducing their size up to 65%. We used uDISCO to image neuronal connections and vasculature from head to toe over 7 cm and to perform unbiased screening of transplanted stem cells within the entire body of adult mice. uDISCO is compatible with diverse labeling methods and archival human tissue, and it can readily be used in various biomedical applications to study organization of large organ systems throughout entire organisms.

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Acknowledgements

This work was supported by the Vascular Dementia Research Foundation, Synergy Excellence Cluster Munich (SyNergy), ERA-Net Neuron (01EW1501A to A.E. and N.P.), and the European Union's Horizon 2020 research and innovation programme (grant agreement no. 666881, SVDs@target, M.D.). A.L. and N.P. were supported by a Marie Curie Intra European Fellowship grant (FP7-PEOPLE-2013-IEF, project no. 625970). We thank M. Hübener and F. Voss (Max Planck Institute of Neurobiology, Munich) for providing mice; D. Trauner and O. Thorn-Seshold for helpful discussions; A. Weingart for illustrations; and C. Hojer, S. Tappan and T. Misgeld for critical reading of the manuscript. C.P. and R.C. are members of the Graduate School of Systemic Neurosciences (GSN), Ludwig Maximilian University of Munich. Human tissues were provided by the brain bank of the Institute of Anatomy, University of Leipzig.

Author information

Author notes

    • Paweł Matryba

    Current address: Department of Molecular and Cellular Neurobiology, Nencki Institute of Experimental Biology of Polish Academy of Sciences, Warsaw, Poland.

    • Chenchen Pan
    • , Ruiyao Cai
    •  & Francesca Paola Quacquarelli

    These authors contributed equally to this work.

Affiliations

  1. Institute for Stroke and Dementia Research, Klinikum der Universität München, Ludwig Maximilians University of Munich (LMU), Munich, Germany.

    • Chenchen Pan
    • , Ruiyao Cai
    • , Francesca Paola Quacquarelli
    • , Alireza Ghasemigharagoz
    • , Athanasios Lourbopoulos
    • , Paweł Matryba
    • , Nikolaus Plesnila
    • , Martin Dichgans
    • , Farida Hellal
    •  & Ali Ertürk
  2. Graduate School of Systemic Neurosciences (GSN), Munich, Germany.

    • Chenchen Pan
    • , Ruiyao Cai
    • , Nikolaus Plesnila
    • , Martin Dichgans
    •  & Ali Ertürk
  3. Munich Cluster for Systems Neurology (SyNergy), Munich, Germany.

    • Nikolaus Plesnila
    • , Martin Dichgans
    • , Farida Hellal
    •  & Ali Ertürk
  4. German Center for Neurodegenerative Diseases (DZNE, Munich), Munich, Germany.

    • Martin Dichgans

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Contributions

A.E. designed and led all aspects of the project. C.P., R.C., and F.P.Q. performed most of the experiments. A.G. performed the image rendering and developed algorithms for data analysis. C.P., R.C., F.P.Q., and A.G. analyzed the data. A.L. interpreted data and performed the BMSC cultures, characterization, and transplantations; F.H. performed virus tracing; P.M. assisted first-clearing experiments; N.P. and M.D. supervised A.L. and F.H., respectively. A.E., C.P., R.C., F.P.Q., and A.G. wrote the paper. All authors edited the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Ali Ertürk.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–30, Supplementary Tables 1 and 2, and Supplementary Protocol

Videos

  1. 1.

    3D visualization of microglia in CX3CR1-EGFP mouse brain

    A ~3 months old CX3CR1-EGFP mouse brain was cleared with uDISCO and imaged with the light-sheet microscopy using the 4x corrected objective. The individual microglia throughout the entire brain are evident.

  2. 2.

    uDISCO clearing of limbs and bones

    Surface reconstruction and 3D maximum intensity projection of a paw with forearm from 4 months old C57BL/6N mouse labeled with Texas Red Dextran after whole-body uDISCO clearing. uDISCO renders the bones fully transparent, allowing the visualization of the vascular details in the entire limbs.

  3. 3.

    Details of vasculature in the forelimb

    3D visualization and orthoslice of the forearm with vasculature labeling in Supplementary Video 2. Fine details of the vasculature are clear throughout the entire scan.

  4. 4.

    3D rendering of vasculature in the entire rat CNS

    The vasculature of a 4-weeks old rat was labeled with Texas Red Dextran. We imaged the entire CNS (the brain plus spinal cord) after whole-body clearing of the rat. Both the large and small vessels are clearly visible throughout the entire scan of ≥13 cm CNS rat tissue.

  5. 5.

    Head-to-limb imaging of the nervous system

    3D visualization of neuronal connections throughout the intact mouse CNS in Figure 4 (GFP-M mouse, 4 months old). The fine details of the neuronal connections are evident from head to the nerves invading the hind limbs.

  6. 6.

    Tracing of the CNS axons over several centimeters

    A single traced axons (red) in the spinal cord of the sample from Figure 4 is shown. The trajectories of individual axons in the entire CNS of the adult mice can be determined. Note that occasional curling of long spinal cord axons does not interfere with the tracing of their entire trajectories. The pseudo-colored traced axon is shown thicker than its actual size for easier visualization.

  7. 7.

    3D visualization of neuronal connections in the intact brain in Figure 4

    A GFP-M mouse brain (4 months old) was cleared with uDISCO and imaged with the light-sheet microscopy. The whole-brain images were obtained using the 4x corrected objective, and high-magnification images in the second part of the video were obtained using the 20x corrected objective.

  8. 8.

    Light-sheet microscopy images of the dendritic spines after uDISCO clearing

    A light-sheet microscopy stack acquired on uDISCO cleared GFP-M mouse brain at 0.5 – 1 mm depth using Zeiss CLARITY objective (25x, NA 1.0), which was directly immersed into BABB-D. The individual dendritic spines are evident throughout the scan.

  9. 9.

    Visualization of Qdot-positive BMSCs IV-injected lung

    The video shows the 3D reconstruction of the densely distributed BMSCs throughout the entire lung. uDISCO rendered the intact lungs fully transparent, allowing to assess the distribution of individual BMSCs.

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DOI

https://doi.org/10.1038/nmeth.3964

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