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Large-Area Fluorescence and Electron Microscopic Correlative Imaging With Multibeam Scanning Electron Microscopy
Shinsuke Shibata1,2, Taro Iseda1,2, Takayuki Mitsuhashi3
1Electron Microscope Laboratory, Keio University School of Medicine, Tokyo, Japan.
Frontiers in Neural Circuits
|May 29, 2019
Summary
Researchers developed a large-area correlative light and electron microscopy (CLEM) technique to visualize molecular localization in large brain sections. This method enables high-resolution imaging of detailed structures in both rodent and primate tissues.
Area of Science:
- Cell Biology
- Microscopy
- Neuroscience
Background:
- Correlative light and electron microscopy (CLEM) advances enable detailed cellular and tissue observation.
- Fluorescence labeling visualizes molecular localization, while tissue clearing expands imaging scope.
- Electron microscopy (EM) currently has limitations in correlative observation area.
Purpose of the Study:
- To develop a large-area CLEM imaging procedure for visualizing specific molecular localization in large-scale EM sections.
- To adapt and detail methods for large-area CLEM applicable to various rodent and primate tissues.
Main Methods:
- Antibody labeling of target molecules followed by sequential fluorescence and gold particle visualization.
- Cryostat sections stained, imaged with light microscopy, postfixed for EM, and silver-enhanced.
- Ultrathin sectioning and multibeam scanning electron microscopy (SEM) for rapid, high-resolution, large-area observation.
Main Results:
- Successful implementation of a large-area CLEM procedure for visualizing molecular localization in mouse and marmoset brain sections.
- Demonstration of multibeam SEM's capability for rapid, high-resolution, large-area imaging in CLEM.
- Detailed methodology provided for applying large-area CLEM across diverse rodent and primate tissues.
Conclusions:
- The developed large-area CLEM procedure significantly expands the observable area for correlative EM studies.
- This technique facilitates detailed structural analysis by combining high-resolution imaging with molecular localization.
- The described methods offer a robust approach for advanced CLEM applications in neuroscience and beyond.
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