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Whole-mouse clearing and imaging at the cellular level with vDISCO

Nature Protocols (2023)Cite this article

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Abstract

Homeostatic and pathological phenomena often affect multiple organs across the whole organism. Tissue clearing methods, together with recent advances in microscopy, have made holistic examinations of biological samples feasible. Here, we report the detailed protocol for nanobody(VHH)-boosted 3D imaging of solvent-cleared organs (vDISCO), a pressure-driven, nanobody-based whole-body immunolabeling and clearing method that renders whole mice transparent in 3 weeks, consistently enhancing the signal of fluorescent proteins, stabilizing them for years. This allows the reliable detection and quantification of fluorescent signal in intact rodents enabling the analysis of an entire body at cellular resolution. Here, we show the high versatility of vDISCO applied to boost the fluorescence signal of genetically expressed reporters and clear multiple dissected organs and tissues, as well as how to image processed samples using multiple fluorescence microscopy systems. The entire protocol is accessible to laboratories with limited expertise in tissue clearing. In addition to its applications in obtaining a whole-mouse neuronal projection map, detecting single-cell metastases in whole mice and identifying previously undescribed anatomical structures, we further show the visualization of the entire mouse lymphatic system, the application for virus tracing and the visualization of all pericytes in the brain. Taken together, our vDISCO pipeline allows systematic and comprehensive studies of cellular phenomena and connectivity in whole bodies.

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Fig. 1: Overview of the vDISCO pipeline.
Fig. 2: vDISCO imaging of a mouse with syngeneic pancreatic cancer.
Fig. 3: Whole-body active-vDISCO setup.
Fig. 4: Whole-body active-vDISCO procedure.
Fig. 5: vDISCO processed Thy1-GFPM brain imaged with Z.1 light-sheet microscope.
Fig. 6: Passive-vDISCO on dissected organs imaged with confocal and light-sheet microscopy.
Fig. 7: Performances of different kinds of nanobodies in passive-vDISCO.
Fig. 8: Prox1-EGFP whole-body scan taken with epifluorescence, light-sheet and confocal microscopy.
Fig. 9: Virus tracing and pericytes in the brain processed by vDISCO.

Data availability

The main data discussed in this protocol are available in the supporting primary research papers (https://www.nature.com/articles/s41593-018-0301-3 and https://www.cell.com/cell/fulltext/S0092-8674(19)31269-3?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS0092867419312693%3Fshowall%3Dtrue). The raw datasets of the main data and of the additional new data shown in this work are too large to be publicly shared but are available for research purposes from the corresponding authors upon reasonable request.

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Acknowledgements

This work was supported by the Vascular Dementia Research Foundation, Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy within the framework of the Munich Cluster for Systems Neurology (EXC 2145 SyNergy, ID 390857198), the ERC Consolidator Grant (AE, 865323), and the Nomis Heart Atlas Project Grant (Nomis Foundation). H.M. is also supported by the China Scholarship Council (CSC) (no. 201806780034). M.M. is supported by the Turkish Ministry of Education for her PhD studies. We thank K. Sleiman, C. Veltkamp and D. Saur for providing the animal bearing pancreatic cancer; M. Voll (Zeiss Microscopy GmbH, Germany) and the Zeiss Microscopy Customer Center Europe for performing the image acquisition with the Lightsheet Z.1; J. Thomas, S. Grade and M. Götz for providing the virus traced sample; J. Shrouder and N. Plesnila for providing the PDGFRb-EGFP mice; A. Ghasemigharagoz, A. Parra-Damas and F.P. Quacquarelli for help during the initial optimization and method development; M. Bralo and B. Forstera for help during the testing of some nanobodies; F. Hellal for critical reading of the manuscript. A.E. and Z.I.K. 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, Zeynep Ilgin Kolabas.

Authors and Affiliations

  1. Institute for Tissue Engineering and Regenerative Medicine (iTERM), Helmholtz Munich, Munich, Germany

    Ruiyao Cai, Zeynep Ilgin Kolabas, Chenchen Pan, Hongcheng Mai, Shan Zhao, Doris Kaltenecker, Muge Molbay, Tzu-lun Ohn, Mihail I. Todorov & Ali Ertürk

  2. Institute for Stroke and Dementia Research, Klinikum der Universität München, Ludwig Maximilian University of Munich, Munich, Germany

    Ruiyao Cai, Zeynep Ilgin Kolabas, Chenchen Pan, Hongcheng Mai, Shan Zhao, Doris Kaltenecker, Muge Molbay, Tzu-lun Ohn, Mihail I. Todorov & Ali Ertürk

  3. Graduate School of Systemic Neurosciences (GSN), Munich, Germany

    Zeynep Ilgin Kolabas

  4. Institute for Diabetes and Cancer, Helmholtz Munich, Munich, Germany

    Doris Kaltenecker

  5. Brain Research Institute, University of Zurich, Zurich, Switzerland

    Fabian F. Voigt & Fritjof Helmchen

  6. Neuroscience Center Zurich, University of Zurich, Zurich, Switzerland

    Fabian F. Voigt & Fritjof Helmchen

  7. Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Brussels, Belgium

    Cécile Vincke & Jo A. Van Ginderachter

  8. Myeloid Cell Immunology Lab, VIB Center for Inflammation Research, Brussels, Belgium

    Cécile Vincke & Jo A. Van Ginderachter

  9. Munich Cluster for Systems Neurology (SyNergy), Munich, Germany

    Ali Ertürk

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  1. Ruiyao Cai
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  2. Zeynep Ilgin Kolabas
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Contributions

A.E. and R.C. initiated the project. R.C. and C.P. developed the original vDISCO method. R.C. designed the experiments. R.C. and Z.I.K. performed most of the experiments. C.P. provided data for the cancer mouse. H.M. and S.Z. provided data for the PDGFRb-EGFP pericyte-labeled brains. F.F.V., M.M., T.-L.O. and F.H. provided mesoSPIM data. C.V. and J.A.V.G. provided the custom-made nanobodies anti-GFP. D.K. contributed to the imaging, and M.I.T helped with the stitching of the Prox1-EGFP sample. R.C. and Z.I.K. supervised the experiments. A.E. supervised the project. R.C. and Z.I.K. wrote the manuscript. All authors edited the manuscript.

Corresponding author

Correspondence to Ali Ertürk.

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Competing interests

A.E., R.C., C.P. and S.Z. have filed a patent related to vDISCO.

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Nature Protocols thanks Nicolas Renier and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Cai, R. et al. Nat. Neurosci. 22, 317–327 (2019): https://doi.org/10.1038/s41593-018-0301-3

Pan, C. et al. Cell 179, 1661–1676.e19 (2019): https://doi.org/10.1016/j.cell.2019.11.013

Extended data

Extended Data Fig. 1 Sample preparation for whole-body active-vDISCO.

a, During the 1× PBS perfusion step of the anesthetized animal, the color of the liver (cyan dashed line) turns yellow as indication of proper drainage of the blood from the body. b, Some cuts in the gut are necessary to flush out the gut content and the feces with a syringe. c, Pictures showing the procedure of skinning the body of the animal using a blade. d, In case the animal is needed with intact skin, commercial hair removal creams may be used as illustrated. The cream should be applied to region of interest and removed with water after 3–5 min. Steps can be repeated until the desired quality of hair removal is achieved. Animal experiments followed European directive 2010/63/EU for animal research, reported according to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) criteria, complied with the ‘3Rs’ measure and were approved by the ethical review board of the government of Upper Bavaria (Regierung von Oberbayern, Munich, Germany) and conformed to institutional guidelines of Klinikum der Universität München/Ludwig Maximilian University of Munich). The severity of the procedure was low.

Extended Data Fig. 2 Whole-body imaging of a sample with intact skin using a modified mesoSPIM light-sheet microscope.

a, Maximum projection of an adult Thy1-GFPM mouse with intact skin imaged with a modified mesoSPIM light-sheet microscope from the ventral side. Autofluorescence excited at 488 nm is shown in green, GFP in magenta. b, Maximum projection of the same sample imaged from the dorsal side (rotated by 180°). c, Layer 5 pyramidal neurons in the brain. d, Peripheral nerves and skin of the forepaw imaged from the lateral direction (90° rotation). e, Detail of the spinal cord and vertebrae. Images in a, b, d and e were taken at 0.9× magnification whereas image in c was imaged at 4× magnification. Animal experiments followed European directive 2010/63/EU for animal research, reported according to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) criteria, complied with the ‘3Rs’ measure and were approved by the ethical review board of the government of Upper Bavaria (Regierung von Oberbayern, Munich, Germany) and conformed to institutional guidelines of Klinikum der Universität München/Ludwig Maximilian University of Munich). The severity of the procedure was low.

Extended Data Fig. 3 Performances of different kinds of nanobodies in passive-vDISCO for Thy1-GFPM line.

a–e, Light-sheet microscopy images of half mouse brains from Thy1-GFPM lines showing the performances of different batches of nanobodies from different sources and companies using standard passive-vDISCO (a–c) and mild passive-vDISCO (d and e). The used nanobody is indicated in the panel title. f, Illustration indicating the anatomic region of the brain (green dashed) that was displayed in a–e. Note that the imaging was taken either on the right or on the left hemisphere; right hemisphere images were flipped to ease the comparison between the different nanobodies. All the results in this figure were similarly observed in at least two independent experiments for each kind of nanobody. Animal experiments followed European directive 2010/63/EU for animal research, reported according to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) criteria, complied with the ‘3Rs’ measure and were approved by the ethical review board of the government of Upper Bavaria (Regierung von Oberbayern, Munich, Germany) and conformed to institutional guidelines of Klinikum der Universität München/Ludwig Maximilian University of Munich). The severity of the procedure was low.

Extended Data Fig. 4 Strategy to make spinal cord straight for passive-vDISCO.

a, Required materials: a plastic Pasteur pipette and some fine needles. The cyan arrowheads indicate the cutting points. b, The plastic Pasteur pipette is then longitudinally cut in half. c, Positioning of the needles to constrain the brain with the spinal cord inside one of the halves of the pipette. d, The whole setting is put into a container such as a 50 ml tube for passive-vDISCO protocol. Animal experiments followed European directive 2010/63/EU for animal research, reported according to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) criteria, complied with the ‘3Rs’ measure and were approved by the ethical review board of the government of Upper Bavaria (Regierung von Oberbayern, Munich, Germany) and conformed to institutional guidelines of Klinikum der Universität München/Ludwig Maximilian University of Munich). The severity of the procedure was low.

Extended Data Fig. 5 Mounting of cleared samples for epifluorescence imaging and inverted confocal imaging.

a–d, Mounting of different samples for AxioZoom epifluorescence imaging: different glass containers used to mount cleared organs (red dashed circle) and slices (magenta boxes) for AxioZoom epifluorescence imaging (a); epifluorescence imaging of dissected organs (red arrowhead) and slices (magenta arrowhead) with the AxioZoom microscope (b and c); epifluorescence imaging of the whole body with the AxioZoom microscope (d). e–g, Mounting of different samples for inverted confocal microscope imaging: a slice (e) and a whole brain (f) are placed onto a glass-bottom dish, then the dish with the lid is positioned onto the stage of the microscope (g). Animal experiments followed European directive 2010/63/EU for animal research, reported according to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) criteria, complied with the ‘3Rs’ measure and were approved by the ethical review board of the government of Upper Bavaria (Regierung von Oberbayern, Munich, Germany) and conformed to institutional guidelines of Klinikum der Universität München/Ludwig Maximilian University of Munich). The severity of the procedure was low.

Extended Data Fig. 6 Mounting of cleared samples for light-sheet imaging.

a–j, Various strategies to mount different samples for LaVision-Miltenyi light-sheet Ultramicroscope II imaging. A whole brain is mounted using the screw system provided by the microscope supplier (a); a slice (d) and a whole brain (e) are mounted using Micro-Fine Ultra needles (b) attached to the sample holder (c–e). To mount a whole head using a flat sample holder (f), a piece of black tape is stuck to the flat surface (g, magenta arrowhead), then by adding super glue (h) and accelerator (i) the head is stabilized onto the tape (j). k–o, Mounting of a whole-body sample for light-sheet imaging using the Blaze microscope: two pieces of black tape are sticked onto the mounting region of the samples holder (k and l, magenta arrowheads), superglue is applied onto the black tapes (m, magenta arrowheads), accelerator is injected into the superglue (n) and the animal is positioned onto the sample holder at the level of the tapes (o). p–r, Strategy to remove air bubbles (p and r, red arrowheads) from the samples (e.g., a whole body) by inserting a fine needle into the bubble (q, cyan arrowhead) and sucking the bubble out with the syringe (r). Animal experiments followed European directive 2010/63/EU for animal research, reported according to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) criteria, complied with the ‘3Rs’ measure and were approved by the ethical review board of the government of Upper Bavaria (Regierung von Oberbayern, Munich, Germany) and conformed to institutional guidelines of Klinikum der Universität München/Ludwig Maximilian University of Munich). The severity of the procedure was low.

Extended Data Fig. 7 Whole-body imaging with a mesoSPIM: setup modifications and sample handling.

a, Overview of the modified mesoSPIM setup: an existing mesoSPIM was modified by replacing the sample XYZ translation stages with stages with larger travel range and by adding a second detection path in the front of the setup. b, Design of the modified detection path with a telecentric detection lens with fixed magnification and a camera with high pixel count. c, The sample was mounted in a custom 40 × 40 × 120 mm3 quartz cuvette. The cuvette was closed with a custom lid that includes a kinematic mount with magnets that attach to the XYZ stages. d, To stabilize the sample inside the cuvette, a 3D-printed crossbar with 40 mm length was inserted above the sample and gently pressed down. e, View of the sample between the mesoSPIM excitation lenses before the front detection path was inserted. Animal experiments followed European directive 2010/63/EU for animal research, reported according to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) criteria, complied with the ‘3Rs’ measure and were approved by the ethical review board of the government of Upper Bavaria (Regierung von Oberbayern, Munich, Germany) and conformed to institutional guidelines of Klinikum der Universität München/Ludwig Maximilian University of Munich). The severity of the procedure was low.

Extended Data Fig. 8 Whole-brain imaging with Zeiss Lightsheet Z.1.

a, The cleared brain sample is glued to the Lightsheet Z.1 rotatable sample holder. b, After mounting the sample holder into the microscope and inserting the imaging chamber containing the clearing solution, the sample is positioned in front of the detection objective. The right position in X, Y, Z and the rotation angle are monitored via the door camera of the microscope. c, The focus plane is adjusted using Near-IR pseudo-bright-field illumination. Animal experiments followed European directive 2010/63/EU for animal research, reported according to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) criteria, complied with the ‘3Rs’ measure and were approved by the ethical review board of the government of Upper Bavaria (Regierung von Oberbayern, Munich, Germany) and conformed to institutional guidelines of Klinikum der Universität München/Ludwig Maximilian University of Munich). The severity of the procedure was low.

Supplementary information

Supplementary Information

Supplementary Figs. 1–3.

Reporting Summary

Supplementary Video 1

Stomach incision and cleaning as a sample preparation step in vDISCO whole-body clearing protocol

Supplementary Video 2

Gut incision and cleaning as a sample preparation step in vDISCO whole-body clearing protocol

Supplementary Video 3

vDISCO clearing protocol pump set-up and demonstration of how to refresh the pumping reference tube slot

Supplementary Video 4

The vDISCO processed Prox1-eGFP mouse with details of lymphatic vessels in the whole body, neurons in the hippocampus and satellite cells in the muscle tissue

Supplementary Video 5

The vDISCO processed brain from an adult Emx1-Cre x ROGT injected with a EnvA-pseudotyped G-deleted rabies virus expressing GFP (SADB1969) that is visible in the neocortex and in the striatum

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Cai, R., Kolabas, Z.I., Pan, C. et al. Whole-mouse clearing and imaging at the cellular level with vDISCO. Nat Protoc (2023). https://doi.org/10.1038/s41596-022-00788-2

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