Analysis of entire transparent rodent bodies after clearing could provide holistic biological information in health and disease, but reliable imaging and quantification of fluorescent protein signals deep inside the tissues has remained a challenge. Here, we developed vDISCO, a pressure-driven, nanobody-based whole-body immunolabeling technology to enhance the signal of fluorescent proteins by up to two orders of magnitude. This allowed us to image and quantify subcellular details through bones, skin and highly autofluorescent tissues of intact transparent mice. For the first time, we visualized whole-body neuronal projections in adult mice. We assessed CNS trauma effects in the whole body and found degeneration of peripheral nerve terminals in the torso. Furthermore, vDISCO revealed short vascular connections between skull marrow and brain meninges, which were filled with immune cells upon stroke. Thus, our new approach enables unbiased comprehensive studies of the interactions between the nervous system and the rest of the body.

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This work was supported by the Vascular Dementia Research Foundation, Synergy Excellence Cluster Munich (SyNergy; EXC 1010), ERA-Net Neuron (01EW1501A; A.E.), Fritz Thyssen Stiftung (reference; A.E.), DFG (reference ER 810/2-1; A.E.), National Institutes of Health (A.E. and M.N.), Helmholtz ICEMED Alliance (A.E.), the Novo Nordisk Foundation (M.N.), the Howard Hughes Medical Institute (B.T.K.) and the Lundbeck Foundation (A.L.R.X. and M.N.). We thank A. Weingart for illustrations, F. Hellal for technical advice and critical reading of the manuscript, and F. P. Quacquarelli and G. Locatelli for help during initial optimization. A.E, C.P., R.C., A.L. and M.I.T. are members of the Graduate School of Systemic Neurosciences at the Ludwig Maximilian University of Munich.

Author information

Author notes

  1. These authors contributed equally: Ruiyao Cai, Chenchen Pan.


  1. Institute for Stroke and Dementia Research, Klinikum der Universität München, Ludwig-Maximilians University Munich, Munich, Germany

    • Ruiyao Cai
    • , Chenchen Pan
    • , Alireza Ghasemigharagoz
    • , Mihail Ivilinov Todorov
    • , Benjamin Förstera
    • , Shan Zhao
    • , Harsharan S. Bhatia
    • , Arnaldo Parra-Damas
    • , Leander Mrowka
    • , Corinne Benakis
    • , Arthur Liesz
    •  & Ali Ertürk
  2. Graduate School of Systemic Neurosciences Munich, Munich, Germany

    • Ruiyao Cai
    • , Chenchen Pan
    • , Mihail Ivilinov Todorov
    • , Arthur Liesz
    •  & Ali Ertürk
  3. Institute of Clinical Neuroimmunology, Klinikum der Universität München, Ludwig-Maximilians University Munich, Munich, Germany

    • Delphine Theodorou
    • , Sabine Liebscher
    •  & Martin Kerschensteiner
  4. Biomedical Center, Ludwig-Maximilians University Munich, Munich, Germany

    • Delphine Theodorou
    • , Sabine Liebscher
    •  & Martin Kerschensteiner
  5. Department of Computer Science and Institute for Advanced Study, Technical University of Munich, Munich, Germany

    • Markus Rempfler
    •  & Bjoern Menze
  6. Center for Translational Neuromedicine, Faculties of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

    • Anna L. R. Xavier
    • , Benjamin T. Kress
    •  & Maiken Nedergaard
  7. Center for Translational Neuromedicine, University of Rochester, New York, NY, USA

    • Benjamin T. Kress
    •  & Maiken Nedergaard
  8. Anatomy Institute, University of Leipzig, Leipzig, Germany

    • Hanno Steinke
    •  & Ingo Bechmann
  9. Munich Cluster for Systems Neurology (SyNergy), Munich, Germany

    • Sabine Liebscher
    • , Arthur Liesz
    • , Martin Kerschensteiner
    •  & Ali Ertürk


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A.E. initiated and led all aspects of the project. R.C. and C.P. developed the method and conducted most of the experiments. R.C. A.G., C.P., H.S.B., M.R., and B.M. analyzed data. M.I.T. stitched and analyzed the whole mouse body scans. A.P.D., B.F., S.Z. and L.M. helped to optimize the protocols. I.B., H.S.B., and S.L. helped to investigate skull–meninges connections. D.T. and M.K. contributed spinal cord injury experiments; C.B. and A.L., MCAO experiments; and A.X., B.K. and M.N., cisterna magna injection experiments. A.E., R.C. and C.P. wrote the paper. All the authors edited the manuscript.

Competing interests

A.E. filed a patent on some of the technologies presented in this work.

Corresponding author

Correspondence to Ali Ertürk.

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figures 1–22 and Supplementary Tables 1 and 2

  2. Reporting Summary

  3. Supplementary Video 1

    vDISCO reveals individual microglia in CX3CR1GFP/+ mouse brain. 3D brain reconstruction of a vDISCO boosted CX3CR1GFP/+ mouse brain, in which microglia express GFP, imaged with 4x objective by light-sheet microscopy. After vDISCO, all labelled microglia became evident, allowing quantification of their numbers in each brain region and assessment of the details of the microglia ramifications. Similar results were observed in 2 independent animals

  4. Supplementary Video 2

    vDISCO reveals whole body neuronal projections in Thy1-GFPM mouse. 3D reconstruction of neuronal projections in a Thy1-GFPM mouse scanned by light-sheet microscopy. The muscles (red) are visualized in autofluorescent channel (blue-green spectra). The bones and internal organs (white) are prominent with PI labeling. The GFP expressing neurons are boosted with nanobody conjugated with Atto 647N and imaged in far-red channel. The overall view of the entire labeled nervous system in the Thy1-GFPM mouse and fine details of neuronal connections are evident throughout the whole body. Comparable labeling and imaging results were achieved in 5 independent animals, whole body reconstruction was done in 2 mice

  5. Supplementary Video 3

    Neuronal projections from spinal cord to right forelimb in Thy1-GFPM mouse. 3D visualization obtained by light-sheet microscopy of neuronal projections from spinal cord to right forelimb of a Thy1-GFPM mouse. The muscles are shown in red, bones in white and the neurons in green. The fine details of axonal extensions and their endings at neuromuscular junctions are visible. Similar results were observed in 2 independent animals

  6. Supplementary Video 4

    vDISCO imaging of neuronal projections in the spinal cord and muscles. The first part of the video is the 2D orthoslicing of the spinal cord of an intact Thy1-GFPM mouse in dorso-ventral orientation. The muscles are shown in red, bones in white and the neurons in green. The details of neuronal cell bodies in ganglia embedded in the spinal cord vertebra and their axonal extensions into the CNS and PNS are visible. In the second part, neuronal connections (green) from spinal cord to muscles are shown in 3D and 2D. Similar results were observed in 2 independent animals

  7. Supplementary Video 5

    vDISCO imaging of CX3CR1GFP/+ mouse with intact skin. The first part of the video is the 3D reconstruction of the inguinal area from a CX3CR1GFP/+ mouse cleared with intact skin, showing inguinal lymph nodes and surrounding tissues (skin and muscles). CX3CR1 GFP+ immune cells are shown in cyan and cell nuclei labeled by PI are shown in magenta. The second part is the 2D orthoslicing visualization of a confocal scan of the same area, showing the subcellular details of CX3CR1 GFP+ cells in the lymph node and around the hair follicles. Similar results were observed in 2 independent animals

  8. Supplementary Video 6

    vDISCO reveals lymphatic vessels of different organs in Prox1-EGFP mouse. After applying vDISCO whole-body labeling of a Prox1-EGFP line mouse, the lungs and intestine were further imaged using high-magnification light-sheet microscopy. Prox1-EGFP lymphatic vessels (green) are evident throughout the tissues. Single experiment

  9. Supplementary Video 7

    Microglia and peripheral immune cells in CX3CR1GFP/+ x CCR2RFP/+ mouse. Multiplexed visualization of CX3CR1GFP/+ x CCR2RFP/+ transgenic mouse head after panoptic imaging with two different nanoboosters (anti-GFP conjugated to Atto647N and anti-RFP conjugated to Atto594N). The CX3CR1 GFP+ microglia cells in the brain parenchyma vs. CCR2 RFP+ peripheral immune cells in the meningeal vessels were clearly visible in 3D reconstruction and 2D orthoslicing. Similar results were observed from 3 independent double transgenic mice

  10. Supplementary Video 8

    Revealing short skull meninges connections (SMCs) in intact CX3CR1GFP/+ mouse heads. 2D orthoslicing of transparent head from the sagittal view of a CX3CR1GFP/+ line mouse imaged by light-sheet microscope. The vasculature was labelled by Lectin (red) and CX3CR1 GFP+ immune cells and microglia cells (green) were boosted by vDISCO. Short skull-meninges connections (SMCs) containing CX3CR1 GFP+ immune cells are evident. Similar results were observed in 3 independent animals

  11. Supplementary Video 9

    Cellular details of SMCs in intact mouse heads. 3D visualization of skull and brain interface in a CX3CR1GFP/+ line mouse imaged by confocal microscope. Vasculature was labelled with lectin (magenta) and CX3CR1 GFP+ cells (green) were boosted by vDISCO. Short vascular connections between skull marrow and meninges at the sagittal sinus and brain interfaces are clearly visualized. We also observed occasional connections between neighbouring skull marrow regions. Note that lectin dye is taken up by phagocytic cells in the skull marrow similar to dextran54. Similar results were observed in 3 independent animals

  12. Supplementary Video 10

    LysM GFP+ immune cells observed in SMCs upon ischemic stroke lesion. 2D orthoslicing of LysM-EGFP mouse head with MCAO. LysM GFP+ neutrophils and monocytes are shown in red and cell nuclei labelled by PI are shown in green. LysM GFP+ cells increased in the lesion site indicating the infiltration of immune cells after MCAO. In addition, many LysM GFP+ cells are observed in the vascular connections to the meninges suggesting that SMCs can be a potential cell trafficking route and play a role in the neuroinflammatory process. Similar results were observed from 4 independent mice per group

  13. Supplementary Video 11

    Widespread inflammation upon SCI assessed by panoptic vDISCO imaging. Panoptic imaging of CD68-EGFP transgenic mouse upon SCI. 2D orthoslicing in horizontal and sagittal views clearly shows the activated immune cells (green) in the muscles, spinal cord roots and meningeal compartments. Vertebra, which become prominent with PI labelling, are shown in white. Similar results were observed from 3 independent mice per group

  14. Supplementary Video 12

    Extensive details of neuronal connections in the mouse brain revealed by vDISCO. A Thy1-GFPM mouse brain was imaged by light-sheet microscopy more than a year after vDISCO. The details of the neuronal structures down to the subcellular level were imaged by Zeiss 20x objective on the light-sheet microscope. Similar results were observed in 2 independent animals

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