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

Author:

Hoffman David P.1ORCID,Shtengel Gleb1ORCID,Xu C. Shan1ORCID,Campbell Kirby R.2ORCID,Freeman Melanie1ORCID,Wang Lei345ORCID,Milkie Daniel E.1ORCID,Pasolli H. Amalia1ORCID,Iyer Nirmala1ORCID,Bogovic John A.1ORCID,Stabley Daniel R.6,Shirinifard Abbas7,Pang Song1,Peale David1ORCID,Schaefer Kathy1ORCID,Pomp Wim345ORCID,Chang Chi-Lun1ORCID,Lippincott-Schwartz Jennifer1ORCID,Kirchhausen Tom1345ORCID,Solecki David J.2,Betzig Eric1891011ORCID,Hess Harald F.1ORCID

Affiliation:

1. Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA.

2. Department of Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA.

3. Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.

4. Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Boston, MA 02115, USA.

5. Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA.

6. Neuroimaging Laboratory, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA.

7. Bioimage Analysis Core, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA.

8. Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.

9. Department of Physics, University of California, Berkeley, CA 94720, USA.

10. Howard Hughes Medical Institute, Berkeley, CA 94720, USA.

11. Helen Wills Neuroscience Institute, Berkeley, CA 94720, USA.

Abstract

Visualizing whole cells at many scales Cells need to compartmentalize thousands of distinct proteins, but the nanoscale spatial relationship of many proteins to overall intracellular ultrastructure remains poorly understood. Correlated light and electron microscopy approaches can help. Hoffman et al. combined cryogenic super-resolution fluorescence microscopy and focused ion beam–milling scanning electron microscopy to visualize protein-ultrastructure relationships in three dimensions across whole cells. The fusion of the two imaging modalities enabled identification and three-dimensional segmentation of morphologically complex structures within the crowded intracellular environment. The researchers observed unexpected relationships within a variety of cell types, including a web-like protein adhesion network between juxtaposed cerebellar granule neurons. Science , this issue p. eaaz5357

Funder

National Institutes of Health

Howard Hughes Medical Institute

National Institute of Neurological Disorders and Stroke

Biogen

St. Jude Children's Research Hospital

American Lebanese Syrian Associated Charities

Publisher

American Association for the Advancement of Science (AAAS)

Subject

Multidisciplinary

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