Three-dimensional SEM, TEM, and STEM for analysis of large-scale biological systems

Radulović, Snježana; Sunkara, Sowmya; Rachel, Reinhard; Leitinger, Gerd

doi:10.1007/s00418-022-02117-w

Cited by 12 publications

(7 citation statements)

References 40 publications

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“…Also, the permeability of reagents and light scattering remain as potential issues for thick samples. Advancements in electron microscopy provide an additional means to obtain biological information at subcellular levels but also suffer from their own limitations ( 45 ). Ultimately, it will be important to combine multiple imaging and non-imaging modalities to address a particular biological question as discussed in a recent review ( 45 ).…”

Section: Discussionmentioning

confidence: 99%

Utilization of commercial collagens for preparing well-differentiated human beta cells for confocal microscopy

Brennecke,

Yang,

Liu

et al. 2023

Front. Endocrinol.

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IntroductionWith technical advances, confocal and super-resolution microscopy have become powerful tools to dissect cellular pathophysiology. Cell attachment to glass surfaces compatible with advanced imaging is critical prerequisite but remains a considerable challenge for human beta cells. Recently, Phelps et al. reported that human beta cells plated on type IV collagen (Col IV) and cultured in neuronal medium preserve beta cell characteristics.MethodsWe examined human islet cells plated on two commercial sources of Col IV (C6745 and C5533) and type V collagen (Col V) for differences in cell morphology by confocal microscopy and secretory function by glucose-stimulated insulin secretion (GSIS). Collagens were authenticated by mass spectrometry and fluorescent collagen-binding adhesion protein CNA35. ResultsAll three preparations allowed attachment of beta cells with high nuclear localization of NKX6.1, indicating a well-differentiated status. All collagen preparations supported robust GSIS. However, the morphology of islet cells differed between the 3 preparations. C5533 showed preferable features as an imaging platform with the greatest cell spread and limited stacking of cells followed by Col V and C6745. A significant difference in attachment behavior of C6745 was attributed to the low collagen contents of this preparation indicating importance of authentication of coating material. Human islet cells plated on C5533 showed dynamic changes in mitochondria and lipid droplets (LDs) in response to an uncoupling agent 2-[2-[4-(trifluoromethoxy)phenyl]hydrazinylidene]-propanedinitrile (FCCP) or high glucose + oleic acid. DiscussionAn authenticated preparation of Col IV provides a simple platform to apply advanced imaging for studies of human islet cell function and morphology.

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Section: Discussionmentioning

confidence: 99%

Utilization of commercial collagens for preparing well-differentiated human beta cells for confocal microscopy

Brennecke,

Yang,

Liu

et al. 2023

Front. Endocrinol.

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“…Because the cTEC network develops meshwork architecture, single section analyses would provide valuable, albeit limited information. We thus obtained 3-dimensional reconstructions at high resolution using 3D OTO-SEM Array Tomography 73 . Backscattered OTO-SEM revealed the ultrastructural details of cTEC cytoreticulum, while it partially or completely enveloped the thymocytes under various stages of their development in the outer cortex (Figs.…”

Section: Ultrastructural Topography Of the Ctec Networkmentioning

confidence: 99%

Morphometric Analysis of the Thymic Epithelial Cell (TEC) Network Using Integrated and Orthogonal Digital Pathology Approaches

Lagou,

Argyris,

Vodopyanov

et al. 2024

Preprint

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The thymus, a central primary lymphoid organ of the immune system, plays a key role in T cell development. Surprisingly, the thymus is quite neglected with regards to standardized pathology approaches and practices for assessing structure and function. Most studies use multispectral flow cytometry to define the dynamic composition of the thymus at the cell population level, but they are limited by lack of contextual insight. This knowledge gap hinders our understanding of various thymic conditions and pathologies, particularly how they affect thymic architecture, and subsequently, immune competence. Here, we introduce a digital pathology pipeline to address these challenges. Our approach can be coupled to analytical algorithms and utilizes rationalized morphometric assessments of thymic tissue, ranging from tissue-wide down to microanatomical and ultrastructural levels. This pipeline enables the quantitative assessment of putative changes and adaptations of thymic structure to stimuli, offering valuable insights into the pathophysiology of thymic disorders. This versatile pipeline can be applied to a wide range of conditions that may directly or indirectly affect thymic structure, ranging from various cytotoxic stimuli inducing acute thymic involution to autoimmune diseases, such as myasthenia gravis. Here, we demonstrate applicability of the method in a mouse model of age-dependent thymic involution, both by confirming established knowledge, and by providing novel insights on intrathymic remodeling in the aged thymus. Our orthogonal pipeline, with its high versatility and depth of analysis, promises to be a valuable and practical toolset for both basic and translational immunology laboratories investigating thymic function and disease.

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“…DNA, RNA, proteins) are too small to be individualized with optical microscopes (Wong and Eleftheriades 2013; Jensen 2013) and consequently, the interior of the cell (and the interior of a nucleus even more) remains largely invisible to the human eye. Alternative solutions are based on molecular-scale techniques like X-ray crystallography (Smyth 2000), NMR microscopy (Reckel et al 2005) or electron microscopy (Radulović et al 2022). By analyzing atom arrangements in molecules, these techniques produce informative views of macromolecular complexity (Nogales and Scheres 2015; Baumeister 2022), but they require complex technical skills and expensive equipment.…”

Section: Introductionmentioning

confidence: 99%

Visual integration of omics data to improve 3D models of fungal chromosomes

Poinsignon

Gallopin

Grognet

et al. 2023

Preprint

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The functions of eukaryotic chromosomes and their spatial architecture in the nucleus are reciprocally dependent. Hi-C experiments are routinely used to study chromosome 3D organization by probing chromatin interactions. Standard representation of the data has relied on contact maps that show the frequency of interactions between parts of the genome. In parallel, it has become easier to build 3D models of the entire genome based on the same Hi-C data, and thus benefit from the methodology and visualization tools developed for structural biology. 3D modeling of entire genomes leverages the understanding of their spatial organization. However, this opportunity for original and insightful modeling is under exploited. In this paper, we show how seeing the spatial organization of chromosomes can bring new perspectives to Hi-C data analysis. We assembled state-of-the-art tools into a workflow that goes from Hi-C raw data to fully annotated 3D models and we re-analysed public Hi-C datasets available for three fungal species. Besides the well-described properties of the spatial organization of their chromosomes (Rabl conformation, hypercoiling and chromosome territories), our 3D models highlighted i) in Saccharomyces cerevisiae, the backbones of the cohesin anchor regions, which were aligned all along the chromosomes, ii) in Schizosaccharomyces pombe, the oscillations of the coiling of chromosome arms throughout the cell cycle and iii) in Neurospora crassa, the massive relocalization of histone marks in mutants of heterochromatin regulators. 3D modeling of the chromosomes brings new opportunities for visual integration. This holistic perspective supports intuition and lays the foundation for building new concepts.

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Three-dimensional SEM, TEM, and STEM for analysis of large-scale biological systems

Cited by 12 publications

References 40 publications

Utilization of commercial collagens for preparing well-differentiated human beta cells for confocal microscopy

Utilization of commercial collagens for preparing well-differentiated human beta cells for confocal microscopy

Morphometric Analysis of the Thymic Epithelial Cell (TEC) Network Using Integrated and Orthogonal Digital Pathology Approaches

Visual integration of omics data to improve 3D models of fungal chromosomes

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