Large-area fluorescence and electron microscopic correlative imaging with multibeam scanning electron microscopy
- 1Electron Microscope Laboratory, School of Medicine, Keio University, Japan
- 2Department of Physiology, Keio University School of Medicine, Japan
- 3Department of Pediatrics, School of Medicine, Keio University, Japan
- 4Central Institute for Experimental Animals, Japan
- 5Tokyo Medical and Dental University, Japan
- 6Department of Molecular and Cellular Biology, Faculty of Arts and Sciences, Harvard University, United States
- 7Laboratory for Marmoset Neural Architecture, RIKEN Center for Brain Science (CBS), Japan
Recent improvements in correlative light and electron microscopy (CLEM) technology have led to dramatic improvements in the ability to observe tissues and cells. Fluorescence labeling has been used to visualize the localization of molecules of interest through immunostaining or genetic modification strategies for the identification of the molecular signatures of biological specimens. Newer technologies such as tissue clearing have expanded the field of observation available for fluorescence labeling; however, the area of correlative observation available for electron microscopy remains restricted.
In this study, we developed a large-area CLEM imaging procedure to show specific molecular localization in large-scale electron microscopy (EM) sections of mouse and marmoset brain. Target molecules were labeled with antibodies and sequentially visualized in cryostat sections using fluorescence and gold particles. Fluorescence images were obtained by light microscopy immediately after antibody staining. Immunostained sections were postfixed for EM, and silver-enhanced sections were dehydrated in a graded ethanol series and embedded in resin. Ultrathin sections for EM were prepared from fully polymerized resin blocks, collected on silicon wafers, and observed by multibeam scanning electron microscopy. Multibeam scanning electron microscopy has made rapid, large-area observation at high resolution possible, paving the way for the analysis of detailed structures using the CLEM approach. Here, we describe detailed methods for large-area CLEM in various tissues of both rodents and primates.
Keywords: Immuno-EM, connectomics, multi-beam SEM, Correlative imaging, CLEM
Received: 16 Nov 2018;
Accepted: 08 Apr 2019.
Edited by:Yoshiyuki Kubota, National Institute for Physiological Sciences (NIPS), Japan
Reviewed by:Yunfeng Hua, School of Medicine, Shanghai Jiao Tong University, China
Naomi Kamasawa, Max Planck Florida Institute for Neuroscience (MPFI), United States
Copyright: © 2019 Shibata, Iseda, Mitsuhashi, Oka, Shindo, Moritoki, Nagai, Otsubo, Inoue, Sasaki, Akazawa, Takahashi, Schalek, Lichtman and Okano. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Dr. Shinsuke Shibata, Electron Microscope Laboratory, School of Medicine, Keio University, Tokyo, Japan, email@example.com
Prof. Hideyuki Okano, Electron Microscope Laboratory, School of Medicine, Keio University, Tokyo, Japan, firstname.lastname@example.org