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Multispectral confocal 3D imaging of intact healthy and tumor tissue using mLSR-3D

Nature Protocols volume 17, pages 3028–3055 (2022)Cite this article

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Abstract

Revealing the 3D composition of intact tissue specimens is essential for understanding cell and organ biology in health and disease. State-of-the-art 3D microscopy techniques aim to capture tissue volumes on an ever-increasing scale, while also retaining sufficient resolution for single-cell analysis. Furthermore, spatial profiling through multi-marker imaging is fast developing, providing more context and better distinction between cell types. Following these lines of technological advance, we here present a protocol based on FUnGI (fructose, urea and glycerol clearing solution for imaging) optical clearing of tissue before multispectral large-scale single-cell resolution 3D (mLSR-3D) imaging, which implements ‘on-the-fly’ linear unmixing of up to eight fluorophores during a single acquisition. Our protocol removes the need for repetitive illumination, thereby allowing larger volumes to be scanned with better image quality in less time, also reducing photo-bleaching and file size. To aid in the design of multiplex antibody panels, we provide a fast and manageable intensity equalization assay with automated analysis to design a combination of markers with balanced intensities suitable for mLSR-3D. We demonstrate effective mLSR-3D imaging of various tissues, including patient-derived organoids and xenografted tumors, and, furthermore, describe an optimized workflow for mLSR-3D imaging of formalin-fixed paraffin-embedded samples. Finally, we provide essential steps for 3D image data processing, including shading correction that does not require pre-acquired shading references and 3D inhomogeneity correction to correct fluorescence artefacts often afflicting 3D datasets. Together, this provides a one-week protocol for eight-fluorescent-marker 3D visualization and exploration of intact tissue of various origins at single-cell resolution.

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Fig. 1: Schematic summary of the mLSR-3D imaging protocol and envisioned applications.
Fig. 2: mLSR-3D equalization assay and FUnGI clearing.
Fig. 3: mLSR-3D imaging of fetal kidney, Wilms tumor, breast cancer xenograft and organoids.
Fig. 4: mLSR-3D imaging of brain tissue and organoids.
Fig. 5: mLSR-3D imaging of deparaffinized FFPE tissue.
Fig. 6: mLSR-3D guided comparative spatio-phenotypic analysis of human brain tumors.

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Data availability

All data described in this protocol are available from the corresponding author upon reasonable request. Representative subsetted datasets and analyses are made publicly available through demos on the STAPL-3D GitHub page (https://github.com/RiosGroup/STAPL3D).

Code availability

All source codes of STAPL-3D are publicly available through GitHub (https://github.com/RiosGroup/STAPL3D).

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Acknowledgements

This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement no. 804412). This work was also financially supported by the Princess Máxima Center for Pediatric Oncology and a St. Baldrick’s Robert J. Arceci International Innovation award to A.C.R.

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Author notes

  1. These authors contributed equally: Ravian L. van Ineveld, Raphaël Collot.

Authors and Affiliations

  1. Princess Máxima Center for Pediatric Oncology, Utrecht, the Netherlands

    Ravian L. van Ineveld, Raphaël Collot, Mario Barrera Román, Anna Pagliaro, Nils Bessler, Hendrikus C. R. Ariese, Michiel Kleinnijenhuis, Marcel Kool, Maria Alieva, Ellen J. Wehrens & Anne C. Rios

  2. Oncode Institute, Utrecht, the Netherlands

    Ravian L. van Ineveld, Raphaël Collot, Mario Barrera Román, Anna Pagliaro, Nils Bessler, Hendrikus C. R. Ariese, Michiel Kleinnijenhuis, Maria Alieva, Ellen J. Wehrens & Anne C. Rios

  3. Hopp Children’s Cancer Center (KiTZ), Heidelberg, Germany

    Marcel Kool

  4. Division of Pediatric Neurooncology, German Cancer Research Center DKFZ and German Cancer Consortium DKTK, Heidelberg, Germany

    Marcel Kool

  5. Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, the Netherlands

    Susana M. Chuva de Sousa Lopes

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Contributions

R.L.v.I. conceived the protocol. R.L.v.I., R.C., M.B.R. and H.C.R.A. performed experiments and analyzed data. A.P. performed FFPE tissue experiments. N.B. and H.C.R.A. performed brain organoid culturing and imaging. M. Kool provided hESC. M. Kleinnijenhuis designed STAPL-3D and performed data analyses. S.M.C.d.S.L. provided human fetal kidney material. M.B.R. made the video. A.C.R. helped design the protocol. R.L.v.I. and R.C. wrote the manuscript. M.B.R., M.A., E.J.W. and A.C.R. co-wrote the manuscript. A.C.R. supervised the project.

Corresponding author

Correspondence to Anne C. Rios.

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

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van Ineveld, R. L. et al. Nat. Biotechnol. 39, 1239–1245 (2021): https://doi.org/10.1038/s41587-021-00926-3

Extended data

Extended Data Fig. 1 Workflow for spectral eight-color mix design with commercially available antibodies.

Emission slot tables serve as an example for marker mix design and present one possible combination of dyes and antibodies specific to brain tissue.

Extended Data Fig. 2 Clearing comparison.

a, Widefield microscopic images of human embryonic kidney tissues uncleared or cleared with FUnGI, Ce3D, CUBIC or iDISCO. Squares represent 1 mm. b, Optical sections at two depths (surface and 100 μm) for all eight channels per condition. Red asterisks indicate lack of specific signal in this channel. Scale bars, 50 μm. c, Quantification of the 10% highest intensity pixels per z-plane per channel for every condition. Channels without sufficient specific signal indicated with a red asterisk were not quantified.

Extended Data Fig. 3 Sequential 32-channel lambda-scan performed on a Leica SP8.

a, Optical section of unmixed eight-color labeled human embryonic kidney. DAPI (not shown), KI67 (aqua), PAX8 (yellow), NCAM1 (magenta), SIX2 (green), CDH1 (red), CDH6 blue) and F-ACTIN (gray). Scale bar, 50 μm. b, Magnification of white inset of a depicting optical sections of combinations of overlay channels for visual inspection. DAPI (gray), F-ACTIN (signal intensity gradient: red-yellow-white) (left), CDH1 (blue), SIX2 (green), NCAM1 (red) (middle), CDH6 (blue), KI67 (green), PAX8 (red) (right). Scale bars, 50 μm. c, Single-channel optical sections of the white inset in a for visual inspection. Scale bars, 50 μm.

Extended Data Fig. 4 Two-photon six-color spectral imaging.

a, 3D rendering of a human pediatric Wilms tumor sample labeled with DAPI (not shown), KI67 (cyan), NCAM1 (blue), SIX2 (green), CDH1 (red) and F-ACTIN (signal intensity gradient: black-red-orange-yellow). b, Wilms tumor dataset labeled with DAPI (not shown), KI67 (cyan), NCAM1 (blue), SIX2 (green), CDH1 (red) and F-ACTIN (gray). Scale bar, 100 μm. c, Single-channel optical sections of the same Wilms tumor dataset. Scale bars, 100 μm.

Supplementary information

Supplementary Information

Supplementary Methods and Supplementary Tables 1 and 2.

Supplementary Movie 1

Summary movie of the mLSR-3D protocol. 0:40–0:50, Optical section z-stack fly-through animation of human embryonic kidney labeled with DAPI (not shown), KI67 (cyan), PAX8 (yellow), NCAM1 (blue), SIX2 (green), CDH1 (red), CDH6 (orange) and F ACTIN (black-red-yellow-white). This movie has been modified from ref. 26. 1:00–1:20, Animation depicting the 3D rendering of breast cancer organoids from Fig. 3d labeled with DAPI (gray), KI67 (green), B-CAT (yellow), CDH1 (red) and K8/18 (blue). 0:00–0:25, Animation depicting the 3D rendering of low-grade glioma from Fig. 6b labeled with DAPI (gray), F ACTIN (black-red-yellow-white), OLIG2 (magenta), TUBB3 (yellow), KI67 (green), GFAP (red), IBA1 (blue) and MAP2 (cyan).

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van Ineveld, R.L., Collot, R., Román, M.B. et al. Multispectral confocal 3D imaging of intact healthy and tumor tissue using mLSR-3D. Nat Protoc 17, 3028–3055 (2022). https://doi.org/10.1038/s41596-022-00739-x

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