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

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

Published: 2022-09-29 DOI: 10.1038/s41596-022-00739-x

single-cell resolution

multispectral confocal imaging

mLSR-3D

tissue clearing

fluorescence unmixing

AI 解读

Extended

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).

Generation of mouse–human chimeric embryos

Genome-wide quantification of transcription factor binding at single-DNA-molecule resolution using methyl-transferase footprinting

Generation of CRISPR–Cas9-mediated genetic knockout human intestinal tissue–derived enteroid lines by lentivirus transduction and single-cell cloning

A puromycin-dependent activity-based sensing probe for histochemical staining of hydrogen peroxide in cells and animal tissues

References

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
Introduction
Materials
Procedure
Troubleshooting
Timing
- Procedure 1: equalization assay
- Procedure 2: general mLSR-3D procedure
Anticipated results
- References
- References
References
ACKNOWLEDGMENTS

Belle, M. et al. Tridimensional visualization and analysis of early human development. Cell 169, 161–173.e12 (2017).
Hannezo, E. et al. A unifying theory of branching morphogenesis. Cell 171, 242–255.e27 (2017).
Rios, A. C., Fu, N. Y., Lindeman, G. J. & Visvader, J. E. In situ identification of bipotent stem cells in the mammary gland. Nature 506, 322–327 (2014).
Scheele, C. L. G. J. et al. Identity and dynamics of mammary stem cells during branching morphogenesis. Nature 542, 313–317 (2017).
Murakami, T. C. et al. A three-dimensional single-cell-resolution whole-brain atlas using CUBIC-X expansion microscopy and tissue clearing. Nat. Neurosci. 21, 625–637 (2018).
Messal, H. A. et al. Tissue curvature and apicobasal mechanical tension imbalance instruct cancer morphogenesis. Nature 566, 126–130 (2019).
Rios, A. C. et al. Intraclonal plasticity in mammary tumors revealed through large-scale single-cell resolution 3D imaging. Cancer Cell 35, 953 (2019).
Brown, M. et al. Lymph node blood vessels provide exit routes for metastatic tumor cell dissemination in mice. Science 359, 1408–1411 (2018).
Almagro, J., Messal, H. A., Thin, M. Z., van Rheenen, J. & Behrens, A. Tissue clearing to examine tumour complexity in three dimensions. Nat. Rev. Cancer 21, 718–730 (2021).
Ertürk, A. et al. Three-dimensional imaging of solvent-cleared organs using 3DISCO. Nat. Protoc. 7, 1983–1995 (2012).
Messal, H. A. et al. Antigen retrieval and clearing for whole-organ immunofluorescence by FLASH. Nat. Protoc. 16, 239–262 (2021).
Dekkers, J. F. et al. High-resolution 3D imaging of fixed and cleared organoids. Nat. Protoc. 14, 1756–1771 (2019).
Bernier-Latmani, J. & Petrova, T. V. High-resolution 3D analysis of mouse small-intestinal stroma. Nat. Protoc. 11, 1617–1629 (2016).
Kusumbe, A. P., Ramasamy, S. K., Starsichova, A. & Adams, R. H. Sample preparation for high-resolution 3D confocal imaging of mouse skeletal tissue. Nat. Protoc. 10, 1904–1914 (2015).
Susaki, E. A. et al. Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging. Nat. Protoc. 10, 1709–1727 (2015).
Tomer, R., Ye, L., Hsueh, B. & Deisseroth, K. Advanced CLARITY for rapid and high-resolution imaging of intact tissues. Nat. Protoc. 9, 1682–1697 (2014).
Renier, N. et al. iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging. Cell 159, 896–910 (2014).
Susaki, E. A. & Ueda, H. R. Whole-body and whole-organ clearing and imaging techniques with single-cell resolution: toward organism-level systems biology in mammals. Cell Chem. Biol. 23, 137–157 (2016).
Tainaka, K., Kuno, A., Kubota, S. I., Murakami, T. & Ueda, H. R. Chemical principles in tissue clearing and staining protocols for whole-body cell profiling. Annu. Rev. Cell Dev. Biol. 32, 713–741 (2016).
Richardson, D. S. & Lichtman, J. W. Clarifying tissue clearing. Cell 162, 246–257 (2015).
Li, W., Germain, R. N. & Gerner, M. Y. High-dimensional cell-level analysis of tissues with Ce3D multiplex volume imaging. Nat. Protoc. 14, 1708–1733 (2019).
Ku, T. et al. Elasticizing tissues for reversible shape transformation and accelerated molecular labeling. Nat. Methods 17, 609–613 (2020).
Goltsev, Y. et al. Deep profiling of mouse splenic architecture with CODEX multiplexed imaging. Cell 174, 968–981.e15 (2018).
Seo, J. et al. PICASSO allows ultra-multiplexed fluorescence imaging of spatially overlapping proteins without reference spectra measurements. Nat. Commun. 13, 2475 (2022).
Murray, E. et al. Simple, scalable proteomic imaging for high-dimensional profiling of intact systems. Cell 163, 1500–1514 (2015).
van Ineveld, R. L. et al. Revealing the spatio-phenotypic patterning of cells in healthy and tumor tissues with mLSR-3D and STAPL-3D. Nat. Biotechnol. 39, 1239–1245 (2021).
Stoltzfus, C. R. et al. CytoMAP: a spatial analysis toolbox reveals features of myeloid cell organization in lymphoid tissues. Cell Rep. 31, 107523 (2020).
Wang, X. et al. Three-dimensional intact-tissue sequencing of single-cell transcriptional states. Science 361, eaat5691 (2018).
Alon, S. et al. Expansion sequencing: spatially precise in situ transcriptomics in intact biological systems. Science 371, eaax2656 (2021).
Valm, A. M. et al. Applying systems-level spectral imaging and analysis to reveal the organelle interactome. Nature 546, 162–167 (2017).
Coutu, D. L., Kokkaliaris, K. D., Kunz, L. & Schroeder, T. Multicolor quantitative confocal imaging cytometry. Nat. Methods 15, 39–46 (2018).
Zimmermann, T., Marrison, J., Hogg, K. & O’Toole, P. Confocal microscopy, methods and protocols. Methods Mol. Biol. 1075, 129–148 (2013).
Susaki, E. A. et al. Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis. Cell 157, 726–739 (2014).
Weiss, K. R., Voigt, F. F., Shepherd, D. P. & Huisken, J. Tutorial: practical considerations for tissue clearing and imaging. Nat. Protoc. 16, 2732–2748 (2021).
Gerner, M. Y., Kastenmuller, W., Ifrim, I., Kabat, J. & Germain, R. N. Histo-cytometry: a method for highly multiplex quantitative tissue imaging analysis applied to dendritic cell subset microanatomy in lymph nodes. Immunity 37, 364–376 (2012).
Gehart, H. et al. Identification of enteroendocrine regulators by real-time single-cell differentiation mapping. Cell 176, 1158–1173.e16 (2019).
van Ineveld, R. L., Ariese, H. C. R., Wehrens, E. J., Dekkers, J. F. & Rios, A. C. Single-cell resolution three-dimensional imaging of intact organoids. J. Vis. Exp. 2020, e60709 (2020).
Calandrini, C. et al. An organoid biobank for childhood kidney cancers that captures disease and tissue heterogeneity. Nat. Commun. 11, 1310 (2020).
Hu, H. et al. Long-term expansion of functional mouse and human hepatocytes as 3D organoids. Cell 175, 1591–1606.e19 (2018).
Post, Y. et al. Snake venom gland organoids. Cell 180, 233–247.e21 (2020).
Schutgens, F. et al. Tubuloids derived from human adult kidney and urine for personalized disease modeling. Nat. Biotechnol. 37, 303–313 (2019).
Lee, S. S.-Y., Bindokas, V. P. & Kron, S. J. Multiplex three-dimensional optical mapping of tumor immune microenvironment. Sci. Rep. 7, 17031 (2017).
Jahr, W., Schmid, B., Schmied, C., Fahrbach, F. O. & Huisken, J. Hyperspectral light sheet microscopy. Nat. Commun. 6, 7990 (2015).
Cutrale, F. et al. Hyperspectral phasor analysis enables multiplexed 5D in vivo imaging. Nat. Methods 14, 149–152 (2017).
Thomson, J. A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998).
Lancaster, M. A. & Knoblich, J. A. Generation of cerebral organoids from human pluripotent stem cells. Nat. Protoc. 9, 2329–2340 (2014).
Taqi, S. A., Sami, S. A., Sami, L. B. & Zaki, S. A. A review of artifacts in histopathology. J. Oral. Maxillofac. Pathol. Jomfp 22, 279–279 (2018).
Kokkat, T. J., Patel, M. S., McGarvey, D., LiVolsi, V. A. & Baloch, Z. W. Archived formalin-fixed paraffin-embedded (FFPE) blocks: a valuable underexploited resource for extraction of DNA, RNA, and protein. Biopreserv. Biobank. 11, 101–106 (2013).
Wong, E. et al. Cut‐point for Ki‐67 proliferation index as a prognostic marker for glioblastoma. Asia Pac. J. Clin. Oncol. 15, 5–9 (2019).
Tavares, C. B., Braga, F. D. C. S. A. G., Sousa, E. B. & de O. Brito, J. N. P. Expression of Ki-67 in low-grade and high-grade astrocytomas. –J. Bras. Neurocirur. 27, 225–230 (2018).