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Correlative three-dimensional super-resolution and block face electron microscopy of whole vitreously frozen cells

View ORCID ProfileDavid P. Hoffman, Gleb Shtengel, View ORCID ProfileC. Shan Xu, View ORCID ProfileKirby R. Campbell, Melanie Freeman, Lei Wang, View ORCID ProfileDaniel E. Milkie, H. Amalia Pasolli, Nirmala Iyer, View ORCID ProfileJohn A. Bogovic, Daniel R. Stabley, View ORCID ProfileAbbas Shirinifard, Song Pang, David Peale, Kathy Schaefer, View ORCID ProfileWim Pomp, View ORCID ProfileChi-Lun Chang, View ORCID ProfileJennifer Lippincott-Schwartz, View ORCID ProfileTom Kirchhausen, View ORCID ProfileDavid J. Solecki, View ORCID ProfileEric Betzig, View ORCID ProfileHarald Hess
doi: https://doi.org/10.1101/773986
David P. Hoffman
1Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA
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Gleb Shtengel
1Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA
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C. Shan Xu
1Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA
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Kirby R. Campbell
2Department of Developmental Neurobiology, St. Jude Children’s Research Hospital, 262 Danny Thomas Pl, Memphis, TN 38105 USA
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Melanie Freeman
1Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA
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Lei Wang
3Department of Cell Biology, Harvard Medical School, 200 Longwood Ave, Boston, MA 02115, USA
4Program in Cellular and Molecular Medicine, Boston Children’s Hospital, 200 Longwood Ave, Boston, MA 02115, USA
5Department of Pediatrics, Harvard Medical School, 200 Longwood Ave, Boston, MA 02115, USA
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Daniel E. Milkie
1Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA
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  • ORCID record for Daniel E. Milkie
H. Amalia Pasolli
1Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA
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Nirmala Iyer
1Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA
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John A. Bogovic
1Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA
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  • ORCID record for John A. Bogovic
Daniel R. Stabley
6Neuroimaging Laboratory, St. Jude Children’s Research Hospital, 262 Danny Thomas Pl, Memphis, TN 38105 USA
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Abbas Shirinifard
7Bioimage Analysis Core, St. Jude Children’s Research Hospital, 262 Danny Thomas Pl, Memphis, TN 38105 USA
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Song Pang
1Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA
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David Peale
1Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA
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Kathy Schaefer
1Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA
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Wim Pomp
3Department of Cell Biology, Harvard Medical School, 200 Longwood Ave, Boston, MA 02115, USA
4Program in Cellular and Molecular Medicine, Boston Children’s Hospital, 200 Longwood Ave, Boston, MA 02115, USA
5Department of Pediatrics, Harvard Medical School, 200 Longwood Ave, Boston, MA 02115, USA
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Chi-Lun Chang
1Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA
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Jennifer Lippincott-Schwartz
1Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA
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Tom Kirchhausen
1Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA
3Department of Cell Biology, Harvard Medical School, 200 Longwood Ave, Boston, MA 02115, USA
4Program in Cellular and Molecular Medicine, Boston Children’s Hospital, 200 Longwood Ave, Boston, MA 02115, USA
5Department of Pediatrics, Harvard Medical School, 200 Longwood Ave, Boston, MA 02115, USA
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David J. Solecki
2Department of Developmental Neurobiology, St. Jude Children’s Research Hospital, 262 Danny Thomas Pl, Memphis, TN 38105 USA
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Eric Betzig
1Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA
8Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA
9Department of Physics, University of California, Berkeley, CA 94720, USA
10Howard Hughes Medical Institute, Berkeley, CA 94720, USA
11Helen Wills Neuroscience Institute, Berkeley, CA 94720, USA
12Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA
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  • For correspondence: hessh@janelia.hhmi.org betzige@janelia.hhmi.org
Harald Hess
1Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA
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  • For correspondence: hessh@janelia.hhmi.org betzige@janelia.hhmi.org
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Abstract

Living cells function through the spatial compartmentalization of thousands of distinct proteins serving a multitude of diverse biochemical needs. Correlative super-resolution (SR) fluorescence and electron microscopy (EM) has emerged as a pathway to directly view nanoscale protein relationships to the underlying global ultrastructure, but has traditionally suffered from tradeoffs of structure preservation, fluorescence retention, resolution, and field of view. We developed a platform for three-dimensional correlative cryogenic SR and focused ion beam milled block-face EM across entire vitreously frozen cells that addresses these issues by preserving native ultrastructure and enabling independent SR and EM workflow optimization. Application to a variety of biological systems revealed a number of unexpected protein-ultrastructure relationships and underscored the value of a comprehensive multimodal view of ultrastructural variability across whole cells.

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Posted September 18, 2019.
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Correlative three-dimensional super-resolution and block face electron microscopy of whole vitreously frozen cells
David P. Hoffman, Gleb Shtengel, C. Shan Xu, Kirby R. Campbell, Melanie Freeman, Lei Wang, Daniel E. Milkie, H. Amalia Pasolli, Nirmala Iyer, John A. Bogovic, Daniel R. Stabley, Abbas Shirinifard, Song Pang, David Peale, Kathy Schaefer, Wim Pomp, Chi-Lun Chang, Jennifer Lippincott-Schwartz, Tom Kirchhausen, David J. Solecki, Eric Betzig, Harald Hess
bioRxiv 773986; doi: https://doi.org/10.1101/773986
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Correlative three-dimensional super-resolution and block face electron microscopy of whole vitreously frozen cells
David P. Hoffman, Gleb Shtengel, C. Shan Xu, Kirby R. Campbell, Melanie Freeman, Lei Wang, Daniel E. Milkie, H. Amalia Pasolli, Nirmala Iyer, John A. Bogovic, Daniel R. Stabley, Abbas Shirinifard, Song Pang, David Peale, Kathy Schaefer, Wim Pomp, Chi-Lun Chang, Jennifer Lippincott-Schwartz, Tom Kirchhausen, David J. Solecki, Eric Betzig, Harald Hess
bioRxiv 773986; doi: https://doi.org/10.1101/773986

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