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Preparation of large biological samples for high-resolution, hierarchical, synchrotron phase-contrast tomography with multimodal imaging compatibility

Nature Protocols volume 18, pages 1441–1461 (2023)Cite this article

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Abstract

Imaging across different scales is essential for understanding healthy organ morphology and pathophysiological changes. The macro- and microscale three-dimensional morphology of large samples, including intact human organs, is possible with X-ray microtomography (using laboratory or synchrotron sources). Preparation of large samples for high-resolution imaging, however, is challenging due to limitations such as sample shrinkage, insufficient contrast, movement of the sample and bubble formation during mounting or scanning. Here, we describe the preparation, stabilization, dehydration and mounting of large soft-tissue samples for X-ray microtomography. We detail the protocol applied to whole human organs and hierarchical phase-contrast tomography at the European Synchrotron Radiation Facility, yet it is applicable to a range of biological samples, including complete organisms. The protocol enhances the contrast when using X-ray imaging, while preventing sample motion during the scan, even with different sample orientations. Bubbles trapped during mounting and those formed during scanning (in the case of synchrotron X-ray imaging) are mitigated by multiple degassing steps. The sample preparation is also compatible with magnetic resonance imaging, computed tomography and histological observation. The sample preparation and mounting require 24–36 d for a large organ such as a whole human brain or heart. The preparation time varies depending on the composition, size and fragility of the tissue. Use of the protocol enables scanning of intact organs with a diameter of 150 mm with a local voxel size of 1 μm. The protocol requires users with expertise in handling human or animal organs, laboratory operation and X-ray imaging.

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Fig. 1: Overview of the sample preparation, stabilization and scanning of a large biological sample.
Fig. 2: HiP-CT images of an intact human heart.
Fig. 3: Example of bubble artifacts.
Fig. 4: Procedures for organ mounting.
Fig. 5: Visualization of an intact human kidney mounted in 70% ethanol crushed agar gel with clinical CT, MRI, sCT and histology.

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Data availability

Image data used to create the figures present in this protocol paper are publicly available from the ESRF data repository (https://human-organ-atlas.esrf.eu) or from the corresponding authors.

Code availability

The sCT data were reconstructed using a custom code written in MATLAB 2017 available on GitHub (https://github.com/HiPCTProject/Tomo_Recon) and the software package PyHST2 (https://software.pan-data.eu/software/74/pyhst2). VGSTUDIO MAX 3.5 (Volume Graphics) was used for volume rendering.

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Acknowledgements

We thank S. Bayat (INSERM) for help during the test phase, P. Masson (LADAF) for dissections of donors’ bodies, H. Reichert (ESRF) and R. Tori for general support of the project and E. Boller, C. Muzelle, R. Homs, C. Jarnias, F. Cianciosi, P. Vieux, P. Cook, L. Capasso and A. Mirone for their help in setup developments and improvements. We also thank R. Engelhardt, A. Muller Brechlin, C. Petzold, N. Kroenke and M. Kuhel for help with histology and autopsies. This project has been made possible in part by grant number 2020-225394 from the Chan Zuckerberg Initiative DAF, an advised fund of Silicon Valley Community Foundation and grant number CZIF2021-006424 from the Chan Zuckerberg Initiative Foundation, The ESRF funding proposals md1252 and md1290, the Royal Academy of Engineering (CiET1819/10). P.D.L. and C.L.W. gratefully acknowledge funding from the MRC (MR/R025673/1). M.A. acknowledges grants from the National Institutes of Health (HL94567 and HL134229). This work was supported by the German Registry of COVID-19 Autopsies (DeRegCOVID, www.DeRegCOVID.ukaachen.de; supported by the Federal Ministry of Health: ZMVI1-2520COR201) and the Federal Ministry of Education and Research as part of the Network of University Medicine (DEFEAT PANDEMIcs, 01KX2021).

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Author notes

  1. These authors contributed equally: J. Brunet, C. L. Walsh.

Authors and Affiliations

  1. Department of Mechanical Engineering, University College London, London, UK

    J. Brunet, C. L. Walsh, S. Marussi & Peter D. Lee

  2. European Synchrotron Radiation Facility, Grenoble, France

    J. Brunet & Paul Tafforeau

  3. Department of Diagnostic and Interventional Radiology, University Hospital Heidelberg, Heidelberg, Germany

    W. L. Wagner

  4. Translational Lung Research Centre Heidelberg (TLRC), German Lung Research Centre (DZL), Heidelberg, Germany

    W. L. Wagner

  5. Laboratoire d’Anatomie des Alpes Françaises (LADAF), Université Grenoble Alpes, Grenoble, France

    A. Bellier

  6. Institute of Pathology, Hannover Medical School, Hannover, Germany

    C. Werlein & D. D. Jonigk

  7. Biomedical Research in End-stage and Obstructive Lung Disease Hannover (BREATH), German Lung Research Centre (DZL), Hannover, Germany

    D. D. Jonigk

  8. Antwerp Surgical Training, Anatomy and Research Centre (ASTARC), University of Antwerp, Antwerp, Belgium

    S. E. Verleden

  9. Institute of Pathology and Molecular Pathology, Helios University Clinic Wuppertal, University of Witten/Herdecke, Wuppertal, Germany

    M. Ackermann

  10. Institute of Functional and Clinical Anatomy, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany

    M. Ackermann

  11. Research Complex at Harwell, Didcot, UK

    Peter D. Lee

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Contributions

P.T., P.D.L., D.D.J., M.A., C.L.W. and W.L.W. conceptualized the project and designed experiments. M.A., C.W., P.T., A.B., S.E.V., C.L.W. and J.B. performed and contributed to autopsies and sample preparation. P.T. designed and built instrumentation and performed HiP-CT imaging. S.M. designed sample holders. P.T. designed and implemented tomographic reconstruction methods. J.B., P.T., C.L.W. and P.D.L. wrote the paper. All authors assisted in reviewing and revising the manuscript.

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Correspondence to J. Brunet, C. L. Walsh, Peter D. Lee or Paul Tafforeau.

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The authors declare no competing interests.

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Nature Protocols thanks Ali Erturk, Stuart Stock and Hiroki Ueda for their contribution to the peer review of this work.

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Key references using this protocol

Walsh, C. L. et al. Nat. Methods 18, 1532–1541 (2021): https://doi.org/10.1038/s41592-021-01317-x

Ackermann, M. et al. Am. J. Respir. Crit. Care Med. 205, 121–125 (2022): https://doi.org/10.1164/rccm.202103-0594IM

Ackermann, M. et al. eBioMedicine 85, 104296 (2022): https://doi.org/10.1016/j.ebiom.2022.104296

Extended data

Extended Data Fig. 1 Challenges faced during the development of the technique and their solutions.

This protocol was developed in an iterative manner overcoming all the different challenges related to soft tissue imaging, dose deposition and local tomography.

Extended Data Fig. 2 Basic information on human organs used in this protocol paper.

The heart, kidney, and brain data are present in the Human Organ Atlas (https://human-organ-atlas.esrf.eu). The liver data are not included in the Human Organ Atlas because of the large number of artifacts present in the images due to bubble formation during scanning. However, the images are available on request from the corresponding authors. The formation of these bubbles occurred because a crash in the beamline software caused the beam to remain in the same position for several hours, exceeding the organ’s dose threshold. Other livers have been scanned without artifacts.

Extended Data Fig. 3 List of human organs and biological samples compatible with this method and the preparation time for each step.

The process described in this protocol paper should work for all major organs, but only the organs listed here have been tested. The maximum degassing times listed here are specific to our vacuum degassing setup and are subject to change depending on the pump, the volume to be pumped, the pumping section, the quantity of gas to be evacuated, and the paths that the gas can take to leave the sample. As such, they should be adapted to each user vacuum setup by looking at the bubbling intensity as explained in Step 4A(ii) of the protocol.

Extended Data Fig. 4 The two types of large leak-proof container in PET in the custom-made container holders.

The left leak-proof container is a Medline Scientific container (cat. no. 129-0592) of 3 L. The right leak-proof container is a Lock & Lock container (cat. no. INL-403) of 2.1 L. Both are inside a custom-made container holder described in Extended Data Fig. 5.

Extended Data Fig. 5 Custom-made container holder drawings and 3D rendering.

Two containers are represented inside the custom-made container holder. The bottom container contains the sample while the top container, referred to as the reference container, is used for the flat-field correction.

Extended Data Fig. 6 Beamline parameters of all the organ scans presented in this paper.

The optimal scanning settings are strongly dependent on the sample and on the beamline setup and equipment. The parameters presented here are given as examples. Quarter acquisition means one scan in half-acquisition plus one annular scan to increase the lateral field of view. Mo = molybdenum.

Supplementary information

Supplementary Video 1

Example of a sample mounted with insufficiently compacted agar, allowing rotation upon a slight movement.

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Brunet, J., Walsh, C.L., Wagner, W.L. et al. Preparation of large biological samples for high-resolution, hierarchical, synchrotron phase-contrast tomography with multimodal imaging compatibility. Nat Protoc 18, 1441–1461 (2023). https://doi.org/10.1038/s41596-023-00804-z

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