Multi-immersion open-top light-sheet microscope for high-throughput imaging of cleared tissues

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Abstract

Recent advances in optical clearing and light-sheet microscopy have provided unprecedented access to structural and molecular information from intact tissues. However, current light-sheet microscopes have imposed constraints on the size, shape, number of specimens, and compatibility with various clearing protocols. Here we present a multi-immersion open-top light-sheet microscope that enables simple mounting of multiple specimens processed with a variety of clearing protocols, which will facilitate wide adoption by preclinical researchers and clinical laboratories. In particular, the open-top geometry provides unsurpassed versatility to interface with a wide range of accessory technologies in the future.

Abstract

Light-sheet microscopes are increasingly used for imaging cleared tissues, but have imposed constraints on sample geometries and protocols. Here the authors present a multi-immersion open-top light-sheet microscope to overcome these limitations and enable high-throughput imaging of samples processed with various clearing protocols.

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Most cited references 24

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Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution.

Bi-Chang Chen, Wesley R Legant, Kai Wang … (2014)

Although fluorescence microscopy provides a crucial window into the physiology of living specimens, many biological processes are too fragile, are too small, or occur too rapidly to see clearly with existing tools. We crafted ultrathin light sheets from two-dimensional optical lattices that allowed us to image three-dimensional (3D) dynamics for hundreds of volumes, often at subsecond intervals, at the diffraction limit and beyond. We applied this to systems spanning four orders of magnitude in space and time, including the diffusion of single transcription factor molecules in stem cell spheroids, the dynamic instability of mitotic microtubules, the immunological synapse, neutrophil motility in a 3D matrix, and embryogenesis in Caenorhabditis elegans and Drosophila melanogaster. The results provide a visceral reminder of the beauty and the complexity of living systems. Copyright © 2014, American Association for the Advancement of Science.

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Three-dimensional imaging of solvent-cleared organs using 3DISCO.

Klaus Becker, Klaus Becker, Nina Jährling … (2013)

The examination of tissue histology by light microscopy is a fundamental tool for investigating the structure and function of organs under normal and disease states. Many current techniques for tissue sectioning, imaging and analysis are time-consuming, and they present major limitations for 3D tissue reconstruction. The introduction of methods to achieve the optical clearing and subsequent light-sheet laser scanning of entire transparent organs without sectioning represents a major advance in the field. We recently developed a highly reproducible and versatile clearing procedure called 3D imaging of solvent-cleared organs, or 3DISCO, which is applicable to diverse tissues including brain, spinal cord, immune organs and tumors. Here we describe a detailed protocol for performing 3DISCO and present its application to various microscopy techniques, including example results from various mouse tissues. The tissue clearing takes as little as 3 h, and imaging can be completed in ∼45 min. 3DISCO is a powerful technique that offers 3D histological views of tissues in a fraction of the time and labor required to complete standard histology studies.

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Shrinkage-mediated imaging of entire organs and organisms using uDISCO.

Chenchen Pan, Ruiyao Cai, Francesca Paola Quacquarelli … (2016)

Recent tissue-clearing approaches have become important alternatives to standard histology approaches. However, light scattering in thick tissues and the size restrictions on samples that can be imaged with standard light-sheet microscopy pose limitations for analyzing large samples such as an entire rodent body. We developed 'ultimate DISCO' (uDISCO) clearing to overcome these limitations in volumetric imaging. uDISCO preserves fluorescent proteins over months and renders intact organs and rodent bodies transparent while reducing their size up to 65%. We used uDISCO to image neuronal connections and vasculature from head to toe over 7 cm and to perform unbiased screening of transplanted stem cells within the entire body of adult mice. uDISCO is compatible with diverse labeling methods and archival human tissue, and it can readily be used in various biomedical applications to study organization of large organ systems throughout entire organisms.

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Author and article information

Contributors

Adam K. Glaser: akglaser@uw.edu

Jonathan T. C. Liu:

ORCID: http://orcid.org/0000-0001-5650-3086

jonliu@uw.edu

Journal

Journal ID (nlm-ta): Nat Commun

Journal ID (iso-abbrev): Nat Commun

Title: Nature Communications

Publisher: Nature Publishing Group UK (London )

ISSN (Electronic): 2041-1723

Publication date (Electronic): 4 July 2019

Publication date PMC-release: 4 July 2019

Publication date Collection: 2019

Volume: 10

Electronic Location Identifier: 2781

Affiliations

[1 ]ISNI 0000000122986657, GRID grid.34477.33, Department of Mechanical Engineering, , University of Washington, ; Seattle, WA 98195 USA

[2 ]ISNI 0000000122986657, GRID grid.34477.33, Department of Pathology, , University of Washington, ; Seattle, WA 98195 USA

[3 ]ISNI 0000000122986657, GRID grid.34477.33, Department of Chemistry, , University of Washington, ; Seattle, WA 98195 USA

[4 ]ISNI 0000000122986657, GRID grid.34477.33, Department of Immunology, , University of Washington, ; Seattle, WA 98109 USA

[5 ]Applied Scientific Instrumentation, Eugene, OR 97402 USA

[6 ]GRID grid.417881.3, Allen Institute for Brain Science, ; Seattle, WA 98109 USA

[7 ]ISNI 0000000122986657, GRID grid.34477.33, Department of Physiology and Biophysics, , University of Washington, ; Seattle, WA 98195 USA

Author information

Linpeng Wei http://orcid.org/0000-0003-3257-7691

Soyoung Kang http://orcid.org/0000-0002-1022-9648

Caleb R. Stoltzfus http://orcid.org/0000-0002-0543-345X

Michael Y. Gerner http://orcid.org/0000-0001-5406-8308

Jonathan T. C. Liu http://orcid.org/0000-0001-5650-3086

Article

Publisher ID: 10534

DOI: 10.1038/s41467-019-10534-0

PMC ID: 6609674

PubMed ID: 31273194

SO-VID: ddeb0542-9d16-41ab-a5f8-8a6184b6a0ae

License:

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

History

Date received : 31 January 2019

Date accepted : 17 May 2019

Funding

Funded by: FundRef https://doi.org/10.13039/100000002, U.S. Department of Health & Human Services | National Institutes of Health (NIH);

Award ID: F32 CA213615

Award ID: DK076169

Award ID: DK115255

Award ID: R01 CA175391

Award ID: R01 DE023497

Award Recipient : Adam K. Glaser Joshua C. Vaughan Jonathan T. C. Liu

Funded by: Prostate Cancer Foundation Young Investigator Award (Reder)

Funded by: FundRef https://doi.org/10.13039/100000005, U.S. Department of Defense (United States Department of Defense);

Award ID: PC170176

Award Recipient : Lawrence D. True Jonathan T. C. Liu

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ScienceOpen disciplines: Uncategorized

Keywords: optical imaging,cancer,neuroscience,systems biology,light-sheet microscopy

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ScienceOpen disciplines: Uncategorized

Keywords: optical imaging, cancer, neuroscience, systems biology, light-sheet microscopy

Multi-immersion open-top light-sheet microscope for high-throughput imaging of cleared tissues

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Most cited references 24

Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution.

Three-dimensional imaging of solvent-cleared organs using 3DISCO.

Shrinkage-mediated imaging of entire organs and organisms using uDISCO.

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