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Morphological and molecular preservation through universal preparation of fresh-frozen tissue samples for multimodal imaging workflows

Nature Protocols volume 19, pages 2685–2711 (2024)Cite this article

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Abstract

The landscape of tissue-based imaging modalities is constantly and rapidly evolving. While formalin-fixed, paraffin-embedded material is still useful for histological imaging, the fixation process irreversibly changes the molecular composition of the sample. Therefore, many imaging approaches require fresh-frozen material to get meaningful results. This is particularly true for molecular imaging techniques such as mass spectrometry imaging, which are widely used to probe the spatial arrangement of the tissue metabolome. As high-quality fresh-frozen tissues are limited in their availability, any sample preparation workflow they are subjected to needs to ensure morphological and molecular preservation of the tissues and be compatible with as many of the established and emerging imaging techniques as possible to obtain the maximum possible insights from the tissues. Here we describe a universal sample preparation workflow, from the initial step of freezing the tissues to the cold embedding in a new hydroxypropyl methylcellulose/polyvinylpyrrolidone-enriched hydrogel and the generation of thin tissue sections for analysis. Moreover, we highlight the optimized storage conditions that limit molecular and morphological degradation of the sections. The protocol is compatible with human and plant tissues and can be easily adapted for the preparation of alternative sample formats (e.g., three-dimensional cell cultures). The integrated workflow is universally compatible with histological tissue analysis, mass spectrometry imaging and imaging mass cytometry, as well as spatial proteomic, genomic and transcriptomic tissue analysis. The protocol can be completed within 4 h and requires minimal prior experience in the preparation of tissue samples for multimodal imaging experiments.

Key points

  • This protocol presents the use of an hydroxypropyl methylcellulose/polyvinylpyrrolidone-rich hydrogel for the cold embedding and sectioning of animal and plant tissues as well as three-dimensional cell cultures.

  • The new embedding material achieves superior morphological preservation of the tissues and makes the specimens compatible with a wide variety of downstream applications, including mass spectrometry imaging, histology and molecular biology methods. The method facilitates the creation of multitissue blocks, limiting batch effects.

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Fig. 1: General snap freezing procedure for fresh tissue samples (Step 1A(i–x)).
Fig. 2: Snap-freezing procedure for encapsulated organs such as kidney and heart (Step 1B(i–xi)).
Fig. 3: Preparation of the HPMC/PVP-based embedding medium (Steps 2–9).
Fig. 4: Embedding of fresh-frozen tissue samples (Steps 10–25).
Fig. 5: Embedding of 3D cell cultures (Steps 43–71).
Fig. 6: Cryosectioning of embedded tissue samples (Steps 72–97).
Fig. 7: Reproducibility evaluation of the optimized embedding protocol and achievable imaging resolution.
Fig. 8: Effects of the embedding media on DNA and RNA extraction.
Fig. 9: IF staining of a cardiac microtissue embedded in HPMC/PVP.

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Data availability

All relevant data are included in the main body of this paper or in the Extended Data. Source data are provided with this paper.

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Acknowledgements

The authors thank the Biotechnology and Biological Sciences Research Council for the case funding for A.D. (BB/N504038/1). The authors also acknowledge the Cancer Research UK Grand Challenge Rosetta Consortium for discussions in support of their research.

Author information

Authors and Affiliations

  1. Imaging and Data analytics, Clinical Pharmacology and Safety Sciences, R&D, AstraZeneca, Cambridge, UK

    Andreas Dannhorn, Lucy Flint, Fei Guo, Alfie Carter, Stewart A. Jones & Richard J. A. Goodwin

  2. Department of Digestion, Metabolism and Reproduction, Sir Alexander Fleming Building, Imperial College London, London, UK

    Andreas Dannhorn, Emine Kazanc & Zoltan Takats

  3. Safety Innovations, Clinical Pharmacology and Safety Sciences, R&D, AstraZeneca, Cambridge, UK

    Fei Guo, Alfie Carter & Andrew R. Hall

  4. The Institute for Cancer Research, London, UK

    George Poulogiannis

  5. Bioscience, Discovery, Oncology R&D, AstraZeneca, Cambridge, UK

    Simon T. Barry

  6. Cancer Research UK Beatson Institute, Glasgow, UK

    Owen J. Sansom

  7. National Centre of Excellence in Mass Spectrometry Imaging (NiCE-MSI), National Physical Laboratory, Teddington, UK

    Josephine Bunch

  8. Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK

    Richard J. A. Goodwin

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  1. Andreas Dannhorn
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Contributions

A.D. and E.K. developed the embedding medium and procedure for tissues. L.F., F.G. and A.R.H. adapted the methodology for the embedding of 3D cell cultures. A.C. and A.R.H. provided the organoids for the appropriate protocol steps. S.A.J. provided the expertise for the freezing procedures of fresh tissue samples. G.P., S.T.B., O.J.S., J.B., Z.T. and R.J.A.G. provided feedback discussion for the methodology. Z.T. and R.J.A.G. secured the funding for the work. All authors reviewed and edited the manuscript drafted by A.D.

Corresponding author

Correspondence to Richard J. A. Goodwin.

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Competing interests

A.D., L.F., F.G., A.C., A.R.H., S.A.J., S.T.B. and R.J.A.G. are full-time salaried employees, own stocks of AstraZeneca and performed this study as part of their regular duties.

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Nature Protocols thanks Junhai Yang, Xiaodong Wang and Miriam Rittel for their contribution to the peer review of this work.

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Related links

Key references using this protocol

Dannhorn, A. et al. Anal. Chem. 92, 11080–11088 (2020): https://doi.org/10.1021/acs.analchem.0c00826

Flint, L. E. et al. Anal. Chem. 92, 12538–12547 (2020): https://doi.org/10.1021/acs.analchem.0c02389

Prag, H. A. et al. Circ. Res. 131, 528–541 (2022): https://doi.org/10.1161/circresaha.121.320717

Vande Voorde, J. et al. Nat. Metab. 5, 1303–1318 (2023): https://doi.org/10.1038/s42255-023-00857-0

Najumudeen, A. K. et al. Nat. Genet. 53, 16–26 (2021): https://doi.org/10.1038/s41588-020-00753-3

Extended data

Extended Data Fig. 1 Impact of choice of cryogen on sample integrity.

a, mouse kidney snap frozen in dry ice chilled isopentane with fracture line through the organ. b, mouse kidney snap frozen in dry ice chilled isopropanol after washing in dry ice chilled isopentane without visible fractures.

Extended Data Fig. 2 Troubleshooting of the freezing of the multi-tissue blocks.

a, Multi-tissue block that was fully frozen before being washed in the isopentane dry ice slurry. b, Multi-tissue block that was not fully frozen before being washed in the isopentane dry ice slurry and that fractured due to pressure building up during rapid freezing. The fracture line is indicated with ▲.

Extended Data Fig. 3 Identification of the sectioning level for 3D cell culture blocks.

a, Top layer of the red-dyed embedding medium. b, Interface layer between the two dyed embedding media in which the 3D cell cultures are positioned and sections of the tissues should be collected. c, tissue-free, blue-dyed base medium after exhaustion of the sample.

Extended Data Fig. 4 Troubleshooting of the sectioning temperature.

a, Section of a multi-tissue block collected at the appropriate temperature without visible fractures of the tissues or sticking to the surfaces of the cryomicrotome. b, Section collected at too low temperature with visible fracturing of the tissues within. c, Section of a multi-tissue block collected at too high temperature with visible damage to the embedding medium and tissue sections.

Extended Data Fig. 5 Troubleshooting of a damaged blade or anti-rolling plate.

a,b, Section of a multi-tissue block with intact blade and the resulting H&E stained tissue section. c,d, Section of the same block collected with a faulty blade and visibly affected tissue morphology on a H&E stained tissue as indicated by ▲. The faulty blade scraped the tissue resulting in bands where the morphological structures were torn apart, rendering them impossible to identify. Scale bars in b and d, 200 µm.

Source data

Source Data Fig. 9

Unprocessed fluorescent microscopy data used to generate Fig. 9.

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Dannhorn, A., Kazanc, E., Flint, L. et al. Morphological and molecular preservation through universal preparation of fresh-frozen tissue samples for multimodal imaging workflows. Nat Protoc 19, 2685–2711 (2024). https://doi.org/10.1038/s41596-024-00987-z

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