Two-color in-resin CLEM of Epon-embedded cells using osmium resistant green and red fluorescent proteins

Abstract In-resin CLEM of Epon embedded samples can greatly simplify the correlation of fluorescent images with electron micrographs. The usefulness of this technique is limited at present by the low number of fluorescent proteins that resist CLEM processing. Additionally, no study has reported the...

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Autores principales:	Isei Tanida, Yoko Furuta, Junji Yamaguchi, Soichiro Kakuta, Juan Alejandro Oliva Trejo, Yasuo Uchiyama
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2020
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/84e16da512444879bfc9d6a0294bd7cd
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spelling	oai:doaj.org-article:84e16da512444879bfc9d6a0294bd7cd2021-12-02T13:34:01ZTwo-color in-resin CLEM of Epon-embedded cells using osmium resistant green and red fluorescent proteins10.1038/s41598-020-78879-x2045-2322https://doaj.org/article/84e16da512444879bfc9d6a0294bd7cd2020-12-01T00:00:00Zhttps://doi.org/10.1038/s41598-020-78879-xhttps://doaj.org/toc/2045-2322Abstract In-resin CLEM of Epon embedded samples can greatly simplify the correlation of fluorescent images with electron micrographs. The usefulness of this technique is limited at present by the low number of fluorescent proteins that resist CLEM processing. Additionally, no study has reported the possibility of two-color in-resin CLEM of Epon embedded cells. In this study, we screened for monomeric green and red fluorescent proteins that resist CLEM processing. We identified mWasabi, CoGFP variant 0, and mCherry2; two green and one red fluorescent proteins as alternatives for in-resin CLEM. We expressed mitochondria-localized mCherry2 and histone H2B tagged with CoGFP variant 0 in cells. Green and red fluorescence was detected in 100 nm-thin sections of the Epon-embedded cells. In the same thin sections, we correlated the fluorescent signals to mitochondria and the nucleus using a scanning electron microscope. Similar results were obtained when endoplasmic reticulum-localized mCherry2 and histone H2B tagged with CoGFP variant 0 were expressed in the cells. Two-color in-resin CLEM of two cytoplasmic organelles, mitochondria and endoplasmic reticulum, was also achieved using mitochondria-localized mCherry2 and endoplasmic reticulum-localized mWasabi. In summary, we report three new fluorescent protein-alternatives suitable for in-resin CLEM of Epon-embedded samples, and achieved Epon-based two-color in-resin CLEM.Isei TanidaYoko FurutaJunji YamaguchiSoichiro KakutaJuan Alejandro Oliva TrejoYasuo UchiyamaNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 10, Iss 1, Pp 1-8 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Isei Tanida Yoko Furuta Junji Yamaguchi Soichiro Kakuta Juan Alejandro Oliva Trejo Yasuo Uchiyama Two-color in-resin CLEM of Epon-embedded cells using osmium resistant green and red fluorescent proteins
description	Abstract In-resin CLEM of Epon embedded samples can greatly simplify the correlation of fluorescent images with electron micrographs. The usefulness of this technique is limited at present by the low number of fluorescent proteins that resist CLEM processing. Additionally, no study has reported the possibility of two-color in-resin CLEM of Epon embedded cells. In this study, we screened for monomeric green and red fluorescent proteins that resist CLEM processing. We identified mWasabi, CoGFP variant 0, and mCherry2; two green and one red fluorescent proteins as alternatives for in-resin CLEM. We expressed mitochondria-localized mCherry2 and histone H2B tagged with CoGFP variant 0 in cells. Green and red fluorescence was detected in 100 nm-thin sections of the Epon-embedded cells. In the same thin sections, we correlated the fluorescent signals to mitochondria and the nucleus using a scanning electron microscope. Similar results were obtained when endoplasmic reticulum-localized mCherry2 and histone H2B tagged with CoGFP variant 0 were expressed in the cells. Two-color in-resin CLEM of two cytoplasmic organelles, mitochondria and endoplasmic reticulum, was also achieved using mitochondria-localized mCherry2 and endoplasmic reticulum-localized mWasabi. In summary, we report three new fluorescent protein-alternatives suitable for in-resin CLEM of Epon-embedded samples, and achieved Epon-based two-color in-resin CLEM.
format	article
author	Isei Tanida Yoko Furuta Junji Yamaguchi Soichiro Kakuta Juan Alejandro Oliva Trejo Yasuo Uchiyama
author_facet	Isei Tanida Yoko Furuta Junji Yamaguchi Soichiro Kakuta Juan Alejandro Oliva Trejo Yasuo Uchiyama
author_sort	Isei Tanida
title	Two-color in-resin CLEM of Epon-embedded cells using osmium resistant green and red fluorescent proteins
title_short	Two-color in-resin CLEM of Epon-embedded cells using osmium resistant green and red fluorescent proteins
title_full	Two-color in-resin CLEM of Epon-embedded cells using osmium resistant green and red fluorescent proteins
title_fullStr	Two-color in-resin CLEM of Epon-embedded cells using osmium resistant green and red fluorescent proteins
title_full_unstemmed	Two-color in-resin CLEM of Epon-embedded cells using osmium resistant green and red fluorescent proteins
title_sort	two-color in-resin clem of epon-embedded cells using osmium resistant green and red fluorescent proteins
publisher	Nature Portfolio
publishDate	2020
url	https://doaj.org/article/84e16da512444879bfc9d6a0294bd7cd
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Two-color in-resin CLEM of Epon-embedded cells using osmium resistant green and red fluorescent proteins

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