Rapid specimen preparation to improve the throughput of electron microscopic volume imaging for three-dimensional analyses of subcellular ultrastructures with serial block-face scanning electron microscopy

2016 ◽  
Vol 49 (3) ◽  
pp. 154-162 ◽  
Author(s):  
Truc Quynh Thai ◽  
Huy Bang Nguyen ◽  
Sei Saitoh ◽  
Bao Wu ◽  
Yurika Saitoh ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Three Dimensional ◽  
Specimen Preparation ◽  
Electron Microscopic ◽  
Volume Imaging ◽  
Face Scanning ◽  
Block Face ◽  
Scanning Electron

scholarly journals Serial Block-Face Scanning Electron Microscopy Reveals That Intercellular Nuclear Migration Occurs in Most Normal Tobacco Male Meiocytes

2021 ◽  
Vol 12 ◽  
Author(s):  
Sergey Mursalimov ◽  
Nobuhiko Ohno ◽  
Mami Matsumoto ◽  
Sergey Bayborodin ◽  
Elena Deineko
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Cell Wall ◽  
Three Dimensional ◽  
Nuclear Migration ◽  
Male Meiosis ◽  
Face Scanning ◽  
Block Face ◽  
Plant Meiosis ◽  
Scanning Electron

Serial block-face scanning electron microscopy (SBF-SEM) was used here to study tobacco male meiosis. Three-dimensional ultrastructural analyses revealed that intercellular nuclear migration (INM) occurs in 90–100% of tobacco meiocytes. At the very beginning of meiosis, every meiocyte connected with neighboring cells by more than 100 channels was capable of INM. At leptotene and zygotene, the nucleus in most tobacco meiocytes approached the cell wall and formed nuclear protuberances (NPs) that crossed the cell wall through the channels and extended into the cytoplasm of a neighboring cell. The separation of NPs from the migrating nuclei and micronuclei formation were not observed. In some cases, the NPs and nuclei of neighboring cells appeared apposed to each other, and the gap between their nuclear membranes became invisible. At pachytene, NPs retracted into their own cells. After that, the INM stopped. We consider INM a normal part of tobacco meiosis, but the reason for such behavior of nuclei is unclear. The results obtained by SBF-SEM suggest that there are still many unexplored features of plant meiosis hidden by limitations of common types of microscopy and that SBF-SEM can turn over a new leaf in plant meiosis research.

scholarly journals Helical fibrillar microstructure of tendon using serial block-face scanning electron microscopy and a mechanical model for interfibrillar load transfer

2019 ◽  
Vol 16 (160) ◽  
pp. 20190547 ◽  
Author(s):  
Babak N. Safa ◽  
John M. Peloquin ◽  
Jessica R. Natriello ◽  
Jeffrey L. Caplan ◽  
Dawn M. Elliott
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Load Transfer ◽  
Three Dimensional ◽  
Helical Structures ◽  
3D Microstructure ◽  
Face Scanning ◽  
Block Face ◽  
Matrix Shear ◽  
Scanning Electron

Tendon's hierarchical structure allows for load transfer between its fibrillar elements at multiple length scales. Tendon microstructure is particularly important, because it includes the cells and their surrounding collagen fibrils, where mechanical interactions can have potentially important physiological and pathological contributions. However, the three-dimensional (3D) microstructure and the mechanisms of load transfer in that length scale are not known. It has been postulated that interfibrillar matrix shear or direct load transfer via the fusion/branching of small fibrils are responsible for load transfer, but the significance of these mechanisms is still unclear. Alternatively, the helical fibrils that occur at the microstructural scale in tendon may also mediate load transfer; however, these structures are not well studied due to the lack of a three-dimensional visualization of tendon microstructure. In this study, we used serial block-face scanning electron microscopy to investigate the 3D microstructure of fibrils in rat tail tendon. We found that tendon fibrils have a complex architecture with many helically wrapped fibrils. We studied the mechanical implications of these helical structures using finite-element modelling and found that frictional contact between helical fibrils can induce load transfer even in the absence of matrix bonding or fibril fusion/branching. This study is significant in that it provides a three-dimensional view of the tendon microstructure and suggests friction between helically wrapped fibrils as a mechanism for load transfer, which is an important aspect of tendon biomechanics.

scholarly journals X-Ray Microscopy as an Approach to Increasing Accuracy and Efficiency of Serial Block-Face Imaging for Correlated Light and Electron Microscopy of Biological Specimens

2014 ◽  
Vol 21 (1) ◽  
pp. 231-238 ◽  
Author(s):  
Eric A. Bushong ◽  
Donald D. Johnson ◽  
Keun-Young Kim ◽  
Masako Terada ◽  
Megumi Hatori ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Light And Electron Microscopy ◽  
Three Dimensional ◽  
Microscopy Study ◽  
Electron Microscopic ◽  
Intense Staining ◽  
X Ray ◽  
Block Face ◽  
Scanning Electron

AbstractThe recently developed three-dimensional electron microscopic (EM) method of serial block-face scanning electron microscopy (SBEM) has rapidly established itself as a powerful imaging approach. Volume EM imaging with this scanning electron microscopy (SEM) method requires intense staining of biological specimens with heavy metals to allow sufficient back-scatter electron signal and also to render specimens sufficiently conductive to control charging artifacts. These more extreme heavy metal staining protocols render specimens light opaque and make it much more difficult to track and identify regions of interest (ROIs) for the SBEM imaging process than for a typical thin section transmission electron microscopy correlative light and electron microscopy study. We present a strategy employing X-ray microscopy (XRM) both for tracking ROIs and for increasing the efficiency of the workflow used for typical projects undertaken with SBEM. XRM was found to reveal an impressive level of detail in tissue heavily stained for SBEM imaging, allowing for the identification of tissue landmarks that can be subsequently used to guide data collection in the SEM. Furthermore, specific labeling of individual cells using diaminobenzidine is detectable in XRM volumes. We demonstrate that tungsten carbide particles or upconverting nanophosphor particles can be used as fiducial markers to further increase the precision and efficiency of SBEM imaging.

scholarly journals High contrast staining for serial block face scanning electron microscopy without uranyl acetate

2017 ◽  
Author(s):  
Adolfo Odriozola ◽  
Jaime Llodrá ◽  
Julika Radecke ◽  
Céline Ruegsegger ◽  
Stefan Tschanz ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Three Dimensional ◽  
Uranyl Acetate ◽  
Radioactive Substance ◽  
Staining Procedure ◽  
Face Scanning ◽  
Block Face ◽  
Scanning Electron

1AbstractSerial block face scanning electron microscopy (SBFSEM) is an increasingly popular method for investigating the three-dimensional ultrastructure of large biological samples. Prior to imaging, samples are typically chemically fixed, stained with osmium and uranyl acetate, and subsequently embedded in resin. The purpose of staining is to provide image contrast and reduce specimen charging under the electron beam, which is detrimental to the quality of imaging. Obtaining, using, and disposing of uranyl acetate is getting increasingly cumbersome in many countries due to new regulations on the handling of radioactive substances. Therefore, we developed an alternative staining procedure that does not rely on the use of uranium or any other radioactive substance. This procedure provides excellent contrast and efficiently reduces specimen charging.

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