A Cryosectioning Technique for the Observation of Intracellular Structures and Immunocytochemistry of Tissues in Atomic Force Microscopy (AFM)

Abstract The use of cryosectioning facilitates the morphological analysis and immunocytochemistry of cells in tissues in atomic force microscopy (AFM). The cantilever can access all parts of a tissue sample in cryosections after the embedding medium (sucrose) has been replaced with phosphate-buffere...

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Autores principales:	Eiji Usukura, Akihiro Narita, Akira Yagi, Nobuaki Sakai, Yoshitsugu Uekusa, Yuka Imaoka, Shuichi Ito, Jiro Usukura
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/8807a19d09354772a50c20bfc434a789
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spelling	oai:doaj.org-article:8807a19d09354772a50c20bfc434a7892021-12-02T11:52:29ZA Cryosectioning Technique for the Observation of Intracellular Structures and Immunocytochemistry of Tissues in Atomic Force Microscopy (AFM)10.1038/s41598-017-06942-12045-2322https://doaj.org/article/8807a19d09354772a50c20bfc434a7892017-07-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-06942-1https://doaj.org/toc/2045-2322Abstract The use of cryosectioning facilitates the morphological analysis and immunocytochemistry of cells in tissues in atomic force microscopy (AFM). The cantilever can access all parts of a tissue sample in cryosections after the embedding medium (sucrose) has been replaced with phosphate-buffered saline (PBS), and this approach has enabled the production of a type of high-resolution image. The images resembled those obtained from freeze-etching replica electron microscopy (EM) rather than from thin-section EM. The AFM images showed disks stacked and enveloped by the cell membrane in rod photoreceptor outer segments (ROS) at EM resolution. In addition, ciliary necklaces on the surface of connecting cilium, three-dimensional architecture of synaptic ribbons, and the surface of the post-synaptic membrane facing the active site were revealed, which were not apparent using thin-section EM. AFM could depict the molecular binding of anti-opsin antibodies conjugated to a secondary fluorescent antibody bound to the disk membrane. The specific localization of the anti-opsin binding sites was verified through correlation with immunofluorescence signals in AFM combined with confocal fluorescence microscope. To prove reproducibility in other tissues besides retina, cryosectioning-AFM was also applied to elucidate molecular organization of sarcomere in a rabbit psoas muscle.Eiji UsukuraAkihiro NaritaAkira YagiNobuaki SakaiYoshitsugu UekusaYuka ImaokaShuichi ItoJiro UsukuraNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-10 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Eiji Usukura Akihiro Narita Akira Yagi Nobuaki Sakai Yoshitsugu Uekusa Yuka Imaoka Shuichi Ito Jiro Usukura A Cryosectioning Technique for the Observation of Intracellular Structures and Immunocytochemistry of Tissues in Atomic Force Microscopy (AFM)
description	Abstract The use of cryosectioning facilitates the morphological analysis and immunocytochemistry of cells in tissues in atomic force microscopy (AFM). The cantilever can access all parts of a tissue sample in cryosections after the embedding medium (sucrose) has been replaced with phosphate-buffered saline (PBS), and this approach has enabled the production of a type of high-resolution image. The images resembled those obtained from freeze-etching replica electron microscopy (EM) rather than from thin-section EM. The AFM images showed disks stacked and enveloped by the cell membrane in rod photoreceptor outer segments (ROS) at EM resolution. In addition, ciliary necklaces on the surface of connecting cilium, three-dimensional architecture of synaptic ribbons, and the surface of the post-synaptic membrane facing the active site were revealed, which were not apparent using thin-section EM. AFM could depict the molecular binding of anti-opsin antibodies conjugated to a secondary fluorescent antibody bound to the disk membrane. The specific localization of the anti-opsin binding sites was verified through correlation with immunofluorescence signals in AFM combined with confocal fluorescence microscope. To prove reproducibility in other tissues besides retina, cryosectioning-AFM was also applied to elucidate molecular organization of sarcomere in a rabbit psoas muscle.
format	article
author	Eiji Usukura Akihiro Narita Akira Yagi Nobuaki Sakai Yoshitsugu Uekusa Yuka Imaoka Shuichi Ito Jiro Usukura
author_facet	Eiji Usukura Akihiro Narita Akira Yagi Nobuaki Sakai Yoshitsugu Uekusa Yuka Imaoka Shuichi Ito Jiro Usukura
author_sort	Eiji Usukura
title	A Cryosectioning Technique for the Observation of Intracellular Structures and Immunocytochemistry of Tissues in Atomic Force Microscopy (AFM)
title_short	A Cryosectioning Technique for the Observation of Intracellular Structures and Immunocytochemistry of Tissues in Atomic Force Microscopy (AFM)
title_full	A Cryosectioning Technique for the Observation of Intracellular Structures and Immunocytochemistry of Tissues in Atomic Force Microscopy (AFM)
title_fullStr	A Cryosectioning Technique for the Observation of Intracellular Structures and Immunocytochemistry of Tissues in Atomic Force Microscopy (AFM)
title_full_unstemmed	A Cryosectioning Technique for the Observation of Intracellular Structures and Immunocytochemistry of Tissues in Atomic Force Microscopy (AFM)
title_sort	cryosectioning technique for the observation of intracellular structures and immunocytochemistry of tissues in atomic force microscopy (afm)
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/8807a19d09354772a50c20bfc434a789
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A Cryosectioning Technique for the Observation of Intracellular Structures and Immunocytochemistry of Tissues in Atomic Force Microscopy (AFM)

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