Development of protocols for the first serial block-face scanning electron microscopy (SBF SEM) studies of bone tissue

Goggin, Patricia; Ho, Elaine M. L.; Gnaegi, Helmut; Searle, Stuart; Oreffo, Richard O.C.; Schneider, Philipp

doi:10.1016/j.bone.2019.115107

Cited by 32 publications

(27 citation statements)

References 74 publications

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“…Serial block‐free scanning electron microscopy (SBF‐SEM) allows three‐dimensional visualization of large sample volumes at ~50 nm resolution 40‐42 . Consistent with the aforementioned two‐dimensional data, compression of microvessels and swelling of surrounding astrocytes were observed in the NVU in the ipsilateral striatum after MCAO under HG condition (Figure 4A).…”

Section: Resultssupporting

confidence: 70%

Ultrastructural studies of the neurovascular unit reveal enhanced endothelial transcytosis in hyperglycemia‐enhanced hemorrhagic transformation after stroke

Zhu

Xue

et al. 2021

CNS Neurosci Ther

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Aims Pre‐existing hyperglycemia (HG) aggravates the breakdown of blood–brain barrier (BBB) and increases the risk of hemorrhagic transformation (HT) after acute ischemic stroke in both animal models and patients. To date, HG‐induced ultrastructural changes of brain microvascular endothelial cells (BMECs) and the mechanisms underlying HG‐enhanced HT after ischemic stroke are poorly understood. Methods We used a mouse model of mild brain ischemia/reperfusion to investigate HG‐induced ultrastructural changes of BMECs that contribute to the impairment of BBB integrity after stroke. Adult male mice received systemic glucose administration 15 min before middle cerebral artery occlusion (MCAO) for 20 min. Ultrastructural characteristics of BMECs were evaluated using two‐dimensional and three‐dimensional electron microscopy and quantitatively analyzed. Results Mice with acute HG had exacerbated BBB disruption and larger brain infarcts compared to mice with normoglycemia (NG) after MCAO and 4 h of reperfusion, as assessed by brain extravasation of the Evans blue dye and microtubule‐associated protein 2 immunostaining. Electron microscopy further revealed that HG mice had more endothelial vesicles in the striatal neurovascular unit than NG mice, which may account for their deterioration of BBB impairment. In contrast with enhanced endothelial transcytosis, paracellular tight junction ultrastructure was not disrupted after this mild ischemia/reperfusion insult or altered upon HG. Consistent with the observed increase of endothelial vesicles, transcytosis‐related proteins caveolin‐1, clathrin, and hypoxia‐inducible factor (HIF)‐1α were upregulated by HG after MCAO and reperfusion. Conclusion Our study provides solid structural evidence to understand the role of endothelial transcytosis in HG‐elicited BBB hyperpermeability. Enhanced transcytosis occurs prior to the physical breakdown of BMECs and is a promising therapeutic target to preserve BBB integrity.

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Section: Resultssupporting

confidence: 70%

Ultrastructural studies of the neurovascular unit reveal enhanced endothelial transcytosis in hyperglycemia‐enhanced hemorrhagic transformation after stroke

Zhu

Xue

et al. 2021

CNS Neurosci Ther

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“…However, structural resolution in ssTEM images is limited using heavy metals on the surfaces of structures, and ssTEM is generally unfit for processing large numbers of samples since many methods lack automation, thus requiring human precision, which may reduce reproducibility and reliability [51,52]. SBF-SEM has resolution ranges of less than 10 nm in the x- and y-axis, which is less than the resolution range of less than 1 nm of ssTEM; however, in the z-axis, the resolution is greater for SBF-SEM at less than 50 nm compared to around 60 nm for ssTEM [15,53]. This makes SEM techniques more effective for 3D reconstruction in many cases.…”

Section: Discussionmentioning

confidence: 99%

“…Despite advances in light microscopy imaging in recent years, EM continues to provide unmatched high resolution ultrastructural detail and 3D visualization [11]. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) are the most common types of electron microscopy used to study organelle ultrastructure [12][13][14][15]. SEM utilizes high-resolution back-scatter detection to provide detailed information of the surface of a sample.…”

mentioning

confidence: 99%

Practices for Measuring 3D Organelle Morphology and Generating Surfaces with Amira

Lopez

Vue

Katti

et al. 2021

Preprint

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Analysis of 3D structures is of paramount importance in cellular biology. Although light microscopy and transmission electron microscopy (TEM) have remained staples for imaging cellular structures, they lack the ability to image in 3D. However, recent technological advances, such as serial block-face scanning electron microscopy (SBF-SEM) and focused ion beam scanning electron microscopy (FIB-SEM), have allowed researchers to observe cellular ultrastructure in 3D. Here, we propose a standardized protocol using the visualization software Amira to quantify organelle morphologies in 3D; this method allows researchers to produce accurate and reproducible measurements of cellular structure characteristics. We demonstrate this applicability by utilizing SBF-SEM and Amira to quantify mitochondria and endoplasmic reticulum (ER) structures.

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“…The endometrial tissue pieces were then stained with respective solutions in order: Osmium/ ferrocyanide fixative for 1 h on ice, Thiocarbohydrazide solution for 20 min, 2% Osmium tetroxide for 30 min, 2% Uranyl acetate for 1 h and Walton’s lead aspartate solution at 60°C for 30 min. Between each incubation and after the Walton’s lead aspartate solution all endometrial pieces had x 5 3 min washes in distilled water (Goggin et al, 2020).…”

Section: Methodsmentioning

confidence: 99%

PAEP and cilia gene splicing changes in endometrial glands during the implantation window in women with recurrent pregnancy loss

Pearson-Farr

Wheway

Jongen

et al. 2021

Preprint

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Endometrial glands are essential for fertility, consisting of ciliated and secretory cells that facilitate a suitable uterine environment for embryo implantation. This study sought to determine whether an endometrial gland specific transcriptome and splicing profile are altered in women with recurrent pregnancy loss. Our data provide a comprehensive catalogue of cilia and PAEP gene isoforms and relative exon usage in endometrial glands. We report a previously unannotated endometrial gland cilia transcript GALNT11 and its susceptibility to exon skipping. Key endometrial receptivity gene transcripts are also reported to change in endometrial glands of women with recurrent pregnancy loss. The endometrial gland cilia and PAEP targets identified in this study could be used to identify a perturbed endometrium, isolate causes of recurrent pregnancy loss and develop targeted therapies in personalised medicine.

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Development of protocols for the first serial block-face scanning electron microscopy (SBF SEM) studies of bone tissue

Cited by 32 publications

References 74 publications

Ultrastructural studies of the neurovascular unit reveal enhanced endothelial transcytosis in hyperglycemia‐enhanced hemorrhagic transformation after stroke

Ultrastructural studies of the neurovascular unit reveal enhanced endothelial transcytosis in hyperglycemia‐enhanced hemorrhagic transformation after stroke

Practices for Measuring 3D Organelle Morphology and Generating Surfaces with Amira

PAEP and cilia gene splicing changes in endometrial glands during the implantation window in women with recurrent pregnancy loss

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