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Ultrastructural visualization of 3D chromatin folding using volume electron microscopy and DNA in situ hybridization.

ABSTRACT: The human genome is extensively folded into 3-dimensional organization. However, the detailed 3D chromatin folding structures have not been fully visualized due to the lack of robust and ultra-resolution imaging capability. Here, we report the development of an electron microscopy method that combines serial block-face scanning electron microscopy with in situ hybridization (3D-EMISH) to visualize 3D chromatin folding at targeted genomic regions with ultra-resolution (5 × 5 × 30 nm in xyz dimensions) that is superior to the current super-resolution by fluorescence light microscopy. We apply 3D-EMISH to human lymphoblastoid cells at a 1.7 Mb segment of the genome and visualize a large number of distinctive 3D chromatin folding structures in ultra-resolution. We further quantitatively characterize the reconstituted chromatin folding structures by identifying sub-domains, and uncover a high level heterogeneity of chromatin folding ultrastructures in individual nuclei, suggestive of extensive dynamic fluidity in 3D chromatin states.

SUBMITTER: Trzaskoma P

PROVIDER: S-EPMC7195386 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Ultrastructural visualization of 3D chromatin folding using volume electron microscopy and DNA in situ hybridization.

Trzaskoma Paweł P Ruszczycki Błażej B Lee Byoungkoo B Pels Katarzyna K KK Krawczyk Katarzyna K Bokota Grzegorz G Szczepankiewicz Andrzej A AA Aaron Jesse J Walczak Agnieszka A Śliwińska Małgorzata A MA Magalska Adriana A Kadlof Michal M Wolny Artur A Parteka Zofia Z Arabasz Sebastian S Kiss-Arabasz Magdalena M Plewczyński Dariusz D Ruan Yijun Y Wilczyński Grzegorz M GM

Nature communications 20200501 1

The human genome is extensively folded into 3-dimensional organization. However, the detailed 3D chromatin folding structures have not been fully visualized due to the lack of robust and ultra-resolution imaging capability. Here, we report the development of an electron microscopy method that combines serial block-face scanning electron microscopy with in situ hybridization (3D-EMISH) to visualize 3D chromatin folding at targeted genomic regions with ultra-resolution (5 × 5 × 30 nm in xyz dimens ...[more]

PMID: 32358536

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Project description:Mainly constituted of glycosaminoglycans and proteoglycans, the glycocalyx is anchored in the plasma membrane, covering, in particular, the extracellular face of the arterial endothelium. Due to its complex three-dimensional (3D) architecture, the glycocalyx interacts with a wide variety of proteins, contributing to vascular permeability, the flow of mechanotransduction, and the modulation of local inflammatory processes. Alterations of glycocalyx structure mediate the endothelial dysfunction and contribute to the aggravation of peripheral vascular diseases. Therefore, the exploration of its ultrastructure becomes a priority to evaluate the degree of injury under physiopathological conditions and to assess the impact of therapeutic approaches. The objective of this study was to develop innovative approaches in electron microscopy to visualize the glycocalyx at the subcellular scale. Intravenous perfusion on rats with a fixing solution containing aldehyde fixatives enriched with lanthanum ions was performed to prepare arterial samples. The addition of lanthanum nitrate in the fixing solution allowed the enhancement of the staining of the glycocalyx for transmission electron microscopy (TEM) and to detect elastic and inelastic scattered electrons, providing complementary qualitative information. The strength of scanning electron microscopy (SEM) was used on resin-embedded serial sections, allowing rapid and efficient large field imaging and previous correlative TEM observations for ultrastructural fine details. To demonstrate the dynamic feature of the glycocalyx, 3D tomography was provided by dual-beam focus-ion-beam-SEM (FIB-SEM). These approaches allowed us to visualize and characterize the ultrastructure of the pulmonary artery glycocalyx under physiological conditions and in a rat pulmonary ischemia-reperfusion model, known to induce endothelial dysfunction. This study demonstrates the feasibility of combined SEM, TEM, and FIB-SEM tomography approaches on the same sample as the multiscale visualization and the identification of structural indicators of arterial endothelial glycocalyx integrity.

Dataset Information

Ultrastructural visualization of 3D chromatin folding using volume electron microscopy and DNA in situ hybridization.

Publications

Ultrastructural visualization of 3D chromatin folding using volume electron microscopy and DNA in situ hybridization.

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