title: High-resolution imaging by scanning electron microscopy of semithin sections in correlation with light microscopy.
abstract: In this study, we introduce scanning electron microscopy (SEM) of semithin resin sections. In this technique, semithin sections were adhered on glass slides, stained with both uranyl acetate and lead citrate, and observed with a backscattered electron detector at a low accelerating voltage. As the specimens are stained in the same manner as conventional transmission electron microscopy (TEM), the contrast of SEM images of semithin sections was similar to TEM images of ultrathin sections. Using this technique, wide areas of semithin sections were also observed by SEM, without the obstruction of grids, which was inevitable for traditional TEM. This study also applied semithin section SEM to correlative light and electron microscopy. Correlative immunofluorescence microscopy and immune-SEM were performed in semithin sections of LR white resin-embedded specimens using a FluoroNanogold-labeled secondary antibody. Because LR white resin is hydrophilic and electron stable, this resin is suitable for immunostaining and SEM observation. Using correlative microscopy, the precise localization of the primary antibody was demonstrated by fluorescence microscopy and SEM. This method has great potential for studies examining the precise localization of molecules, including Golgi- and ER-associated proteins, in correlation with LM and SEM.
authors.affiliation: Division of Microscopic Anatomy and Bio-imaging, Niigata University Graduate School of Medical and Dental Sciences, Niigata 951-8510, Japan Department of Microscopic Anatomy and Cell Biology, Asahikawa Medical University, Asahikawa 078-8510, Hokkaido, Japan dk1224@med.niigata-u.ac.jp.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Koga
authors.affiliation: Division of Microscopic Anatomy and Bio-imaging, Niigata University Graduate School of Medical and Dental Sciences, Niigata 951-8510, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Kusumi
authors.affiliation: Department of Otolaryngology-Head and Neck Surgery, Faculty of Medicine, Niigata University, Niigata 951-8510, Japan.
authors.forename: Ryusuke
authors.initials: R
authors.lastname: Shodo
authors.affiliation: Hitachi High-Technologies Corporation Science and Medical Systems Business Group, Kawasaki 213-0012, Japan.
authors.forename: Yukari
authors.initials: Y
authors.lastname: Dan
authors.affiliation: Division of Microscopic Anatomy and Bio-imaging, Niigata University Graduate School of Medical and Dental Sciences, Niigata 951-8510, Japan.
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Ushiki
mesh_terms: D000818:Animals; D005455:Fluorescent Antibody Technique; D005727:Ganglia, Spinal / Q000166:cytology*; D008297:Male; D008855:Microscopy, Electron, Scanning / Q000379:methods*; D046529:Microscopy, Electron, Transmission / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods*; D008867:Microtomy / Q000379:methods*; D009942:Organometallic Compounds / Q000737:chemistry; D009992:Osmium / Q000737:chemistry; D051381:Rats; D017208:Rats, Wistar; D013194:Staining and Labeling / Q000379:methods; D014021:Tissue Preservation / Q000379:methods
chemical_list: D009942:Organometallic Compounds; C005460:uranyl acetate; D009992:Osmium
keywords: FluoroNanogold; backscattered electron; correlative light and electron microscopy; immune-SEM; scanning electron microscopy; semithin section