Serial Block-Face Scanning Electron Microscopy to Reconstruct Three-Dimensional Tissue Nanostructure
Open accessAbstract
Three-dimensional (3D) structural information on many length scales is of central importance in biological research. Excellent methods exist to obtain structures of molecules at atomic, organelles at electron microscopic, and tissue at light-microscopic resolution. A gap exists, however, when 3D tissue structure needs to be reconstructed over hundreds of micrometers with a resolution sufficient to follow the thinnest cellular processes and to identify small organelles such as synaptic vesicles. Such 3D data are, however, essential to understand cellular networks that, particularly in the nervous system, need to be completely reconstructed throughout a substantial spatial volume. Here we demonstrate that datasets meeting these requirements can be obtained by automated block-face imaging combined with serial sectioning inside the chamber of a scanning electron microscope. Backscattering contrast is used to visualize the heavy-metal staining of tissue prepared using techniques that are routine for transmission electron microscopy. Low-vacuum (20-60 Pa H(2)O) conditions prevent charging of the uncoated block face. The resolution is sufficient to trace even the thinnest axons and to identify synapses. Stacks of several hundred sections, 50-70 nm thick, have been obtained at a lateral position jitter of typically under 10 nm. This opens the possibility of automatically obtaining the electron-microscope-level 3D datasets needed to completely reconstruct the connectivity of neuronal circuits.
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The hemibrain was reconstructed from images acquired by FIB-SEM, a form of block face EM.
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Self-citation
- Authors
- Denk, W., & Horstmann, H.
- Affiliations
- Max Planck Society (DE)
- Corresponding author
- Denk, Winfried
- ORCID
- Denk, Winfried
- Venue
- PLoS Biology
- Publisher
- Public Library of Science (PLoS)
- Published
- 2004-10-19
- Volume
- 2
- Issue
- 11
- Pages
- e329
- ISSN
- 1544-9173 (ISSN-L)
- 1545-7885
- PMC
- PMC524270
- OpenAlex
- W2038665424
- Cited by
- 1,644
- References
- 44
- Citations by year
- 2026: 53 · 2025: 56 · 2024: 83 · 2023: 88 · 2022: 63 · 2021: 108 · 2020: 125 · 2019: 118 · 2018: 108 · 2017: 95 · 2016: 111 · 2015: 107 · 2014: 122 · 2013: 104 · 2012: 72
- FWCI
- 12.85
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- PLoS Biology (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- RePEc: Research Papers in Economics (submittedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Max Planck Institute for Plasma Physics (submittedVersion)
- MPG.PuRe (Max Planck Society) (submittedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Structural Biology › Advanced Electron Microscopy Techniques and Applications
- Physical Sciences › Materials Science › Surfaces, Coatings and Films › Electron and X-Ray Spectroscopy Techniques
- Physical Sciences › Engineering › Biomedical Engineering › Advanced Materials Characterization Techniques
- Keywords
- serial block-face scanning electron microscopy, heavy metal staining, synapses, axon tracing, synaptic vesicles
- MeSH terms
- Animals, Brain, Data Interpretation, Statistical, Electrons, Image Processing, Computer-Assisted, Microscopy, Electron, Scanning, Nerve Net, Nervous System, Neurons, Scattering, Radiation, Synapses, Imaging, Three-Dimensional, Nanostructures, Mice
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