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

ABSTRACT: There is an unmet need for a high-resolution three-dimensional (3D) technique to simultaneously image osteocytes and the matrix in which these cells reside. In serial block-face scanning electron microscopy (SBF SEM), an ultramicrotome mounted within the vacuum chamber of a microscope repeatedly sections a resin-embedded block of tissue. Backscattered electron scans of the block face provide a stack of high-resolution two-dimensional images, which can be used to visualise and quantify cells and organelles in 3D. High-resolution 3D images of biological tissues from SBF SEM have been exploited considerably to date in the neuroscience field. However, non-brain samples, in particular hard biological tissues, have appeared more challenging to image by SBF SEM due to the difficulties of sectioning and rendering the samples conductive. We have developed and propose protocols for bone tissue preparation using SBF SEM, for imaging simultaneously soft and hard bone tissue components in 3D. We review the state of the art in high-resolution imaging of osteocytes, provide a historical perspective of SBF SEM, and we present first SBF SEM proof-of-concept studies for murine and human tissue. The application of SBF SEM to hard tissues will facilitate qualitative and quantitative 3D studies of tissue microstructure and ultrastructure in bone development, ageing and pathologies such as osteoporosis and osteoarthritis.

SUBMITTER: Goggin P

PROVIDER: S-EPMC6961117 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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

Goggin Patricia P Ho Elaine M L EML Gnaegi Helmut H Searle Stuart S Oreffo Richard O C ROC Schneider Philipp P

Bone 20191024

There is an unmet need for a high-resolution three-dimensional (3D) technique to simultaneously image osteocytes and the matrix in which these cells reside. In serial block-face scanning electron microscopy (SBF SEM), an ultramicrotome mounted within the vacuum chamber of a microscope repeatedly sections a resin-embedded block of tissue. Backscattered electron scans of the block face provide a stack of high-resolution two-dimensional images, which can be used to visualise and quantify cells and ...[more]

PMID: 31669251

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Project description:Serial block face scanning electron microscopy (SBF-SEM) is a relatively new technique that allows the acquisition of serially sectioned, imaged and digitally aligned ultrastructural data. There is a wealth of information that can be obtained from the resulting image stacks but this presents a new challenge for researchers - how to computationally analyse and make best use of the large datasets produced. One approach is to reconstruct structures and features of interest in 3D. However, the software programmes can appear overwhelming, time-consuming and not intuitive for those new to image analysis. There are a limited number of published articles that provide sufficient detail on how to do this type of reconstruction. Therefore, the aim of this paper is to provide a detailed step-by-step protocol, accompanied by tutorial videos, for several types of analysis programmes that can be used on raw SBF-SEM data, although there are more options available than can be covered here. To showcase the programmes, datasets of skeletal muscle from foetal and adult guinea pigs are initially used with procedures subsequently applied to guinea pig cardiac tissue and locust brain. The tissue is processed using the heavy metal protocol developed specifically for SBF-SEM. Trimmed resin blocks are placed into a Zeiss Sigma SEM incorporating the Gatan 3View and the resulting image stacks are analysed in three different programmes, Fiji, Amira and MIB, using a range of tools available for segmentation. The results from the image analysis comparison show that the analysis tools are often more suited to a particular type of structure. For example, larger structures, such as nuclei and cells, can be segmented using interpolation, which speeds up analysis; single contrast structures, such as the nucleolus, can be segmented using the contrast-based thresholding tools. Knowing the nature of the tissue and its specific structures (complexity, contrast, if there are distinct membranes, size) will help to determine the best method for reconstruction and thus maximize informative output from valuable tissue.

Project description:The cornea is the most highly innervated tissue in the body. It is generally accepted that corneal stromal nerves penetrate the epithelial basal lamina giving rise to intra-epithelial nerves. During the course of a study wherein we imaged corneal nerves in mice, we observed a novel neuronal-epithelial cell interaction whereby nerves approaching the epithelium in the cornea fused with basal epithelial cells, such that their plasma membranes were continuous and the neuronal axoplasm freely abutted the epithelial cytoplasm. In this study we sought to determine the frequency, distribution, and morphological profile of neuronal-epithelial cell fusion events within the cornea. Serial electron microscopy images were obtained from the anterior stroma in the paralimbus and central cornea of 8-10 week old C57BL/6J mice. We found evidence of a novel alternative behavior involving a neuronal-epithelial interaction whereby 42.8% of central corneal nerve bundles approaching the epithelium contain axons that fuse with basal epithelial cells. The average surface-to-volume ratio of a penetrating nerve was 3.32, while the average fusing nerve was smaller at 1.39 (p ? 0.0001). Despite this, both neuronal-epithelial cell interactions involve similarly sized discontinuities in the basal lamina. In order to verify the plasma membrane continuity between fused neurons and epithelial cells we used the lipophilic membrane tracer DiI. The majority of corneal nerves were labeled with DiI after application to the trigeminal ganglion and, consistent with our ultrastructural observations, fusion sites recognized as DiI-labeled basal epithelial cells were located at points of stromal nerve termination. These studies provide evidence that neuronal-epithelial cell fusion is a cell-cell interaction that occurs primarily in the central cornea, and fusing nerve bundles are morphologically distinct from penetrating nerve bundles. This is, to our knowledge, the first description of neuronal-epithelial cell fusion in the literature adding a new level of complexity to the current understanding of corneal innervation.

Dataset Information

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

Publications

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

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