scholarly journals Dynamic-ultrastructural cell volume (3D) correlative microscopy facilitated by intracellular fluorescent nanodiamonds as multi-modal probes

2019 ◽  
Author(s):  
Neeraj Prabhakar ◽  
Ilya Belevich ◽  
Markus Peurla ◽  
Xavier Heiligenstein ◽  
Huan-Cheng Chang ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Volume ◽  
Light And Electron Microscopy ◽  
Three Dimensional ◽  
Correlative Microscopy ◽  
Straightforward Method ◽  
Functional Aspects ◽  
A Cell ◽  
Block Face ◽  
Fluorescent Nanodiamonds

ABSTRACTThree-dimensional correlative light and electron microscopy (3D CLEM) are attaining popularity as a potential technique to explore the functional aspects of a cell together with high-resolution ultrastructural details across the cell volume. In order to perform such a 3D CLEM experiment, there is an imperative requirement for multi-modal probes that are both fluorescent and electron-dense. These multi-modal probes will serve as landmarks in matching up the large full cell volume datasets acquired by different imaging modalities. Fluorescent nanodiamonds (FNDs) are a unique nanosized, fluorescent, and electron-dense material from the nanocarbon family. We hereby propose a novel and straightforward method for executing 3D CLEM using FNDs as multi-modal landmarks. We demonstrate that FNDs is biocompatible and easily identified both in living cell fluorescence imaging and in serial block-face scanning electron microscopy (SB-EM). We illustrate the 3D CLEM method by registering multi-modal datasets.

scholarly journals Cell Volume (3D) Correlative Microscopy Facilitated by Intracellular Fluorescent Nanodiamonds as Multi-Modal Probes

Nanomaterials ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 14
Author(s):  
Neeraj Prabhakar ◽  
Ilya Belevich ◽  
Markus Peurla ◽  
Xavier Heiligenstein ◽  
Huan-Cheng Chang ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Volume ◽  
Light And Electron Microscopy ◽  
Three Dimensional ◽  
Correlative Microscopy ◽  
Straightforward Method ◽  
Functional Aspects ◽  
A Cell ◽  
Block Face ◽  
Fluorescent Nanodiamonds

Three-dimensional correlative light and electron microscopy (3D CLEM) is attaining popularity as a potential technique to explore the functional aspects of a cell together with high-resolution ultrastructural details across the cell volume. To perform such a 3D CLEM experiment, there is an imperative requirement for multi-modal probes that are both fluorescent and electron-dense. These multi-modal probes will serve as landmarks in matching up the large full cell volume datasets acquired by different imaging modalities. Fluorescent nanodiamonds (FNDs) are a unique nanosized, fluorescent, and electron-dense material from the nanocarbon family. We hereby propose a novel and straightforward method for executing 3D CLEM using FNDs as multi-modal landmarks. We demonstrate that FND is biocompatible and is easily identified both in living cell fluorescence imaging and in serial block-face scanning electron microscopy (SB-EM). We illustrate the method by registering multi-modal datasets.

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 Ultrastructural Characterization of Flashing Mitochondria

Contact ◽  
2018 ◽  
Vol 1 ◽  
pp. 251525641880142
Author(s):  
Manon Rosselin ◽  
Paula Nunes-Hasler ◽  
Nicolas Demaurex
Keyword(s):  
Electron Microscopy ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Light And Electron Microscopy ◽  
Three Dimensional ◽  
Volume Ratio ◽  
Tubular Structures ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Membrane Contact

Mitochondria undergo spontaneous transient elevations in matrix pH associated with drops in mitochondrial membrane potential. These mitopHlashes require a functional respiratory chain and the profusion protein optic atrophy 1, but their mechanistic basis is unclear. To gain insight on the origin of these dynamic events, we resolved the ultrastructure of flashing mitochondria by correlative light and electron microscopy. HeLa cells expressing the matrix-targeted pH probe mitoSypHer were screened for mitopHlashes and fixed immediately after the occurrence of a flashing event. The cells were then processed for imaging by serial block face scanning electron microscopy using a focused ion beam to generate ∼1,200 slices of 10 nm thickness from a 28 µm × 15 µm cellular volume. Correlation of live/fixed fluorescence and electron microscopy images allowed the unambiguous identification of flashing and nonflashing mitochondria. Three-dimensional reconstruction and surface mapping revealed that each tomogram contained two flashing mitochondria of unequal sizes, one being much larger than the average mitochondrial volume. Flashing mitochondria were 10-fold larger than silent mitochondria but with a surface to volume ratio and a cristae volume similar to nonflashing mitochondria. Flashing mitochondria were connected by tubular structures, formed more membrane contact sites, and a constriction was observed at a junction between a flashing mitochondrion and a nonflashing mitochondrion. These data indicate that flashing mitochondria are structurally preserved and bioenergetically competent but form numerous membrane contact sites and are connected by tubular structures, consistent with our earlier suggestion that mitopHlashes might be triggered by the opening of fusion pores between contiguous mitochondria.

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 Dynamics of in vivo ASC speck formation

2017 ◽  
Vol 216 (9) ◽  
pp. 2891-2909 ◽  
Author(s):  
Paola Kuri ◽  
Nicole L. Schieber ◽  
Thomas Thumberger ◽  
Joachim Wittbrodt ◽  
Yannick Schwab ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Death ◽  
Light And Electron Microscopy ◽  
Three Dimensional ◽  
Inflammasome Activation ◽  
Caspase Activation ◽  
Endogenous Gene ◽  
Pyrin Domain ◽  
Effector Caspase

Activated danger or pathogen sensors trigger assembly of the inflammasome adaptor ASC into specks, large signaling platforms considered hallmarks of inflammasome activation. Because a lack of in vivo tools has prevented the study of endogenous ASC dynamics, we generated a live ASC reporter through CRISPR/Cas9 tagging of the endogenous gene in zebrafish. We see strong ASC expression in the skin and other epithelia that act as barriers to insult. A toxic stimulus triggered speck formation and rapid pyroptosis in keratinocytes in vivo. Macrophages engulfed and digested that speck-containing, pyroptotic debris. A three-dimensional, ultrastructural reconstruction, based on correlative light and electron microscopy of the in vivo assembled specks revealed a compact network of highly intercrossed filaments, whereas pyrin domain (PYD) or caspase activation and recruitment domain alone formed filamentous aggregates. The effector caspase is recruited through PYD, whose overexpression induced pyroptosis but only after substantial delay. Therefore, formation of a single, compact speck and rapid cell-death induction in vivo requires a full-length ASC.

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 ON THE PLASTIDS, MITOCHONDRIA, AND OTHER CELL CONSTITUENTS DURING OÖGENESIS OF A PLANT

1966 ◽  
Vol 28 (3) ◽  
pp. 527-543 ◽  
Author(s):  
Lothar Diers
Keyword(s):  
Electron Microscopy ◽  
Cell Volume ◽  
Mother Cell ◽  
Developmental Stages ◽  
Three Dimensional ◽  
Egg Cell ◽  
Lipid Bodies ◽  
Growth Phases ◽  
Quantitative Investigation ◽  
Nucleus Volume

In the liverwort Sphaerocarpus donnellii Aust., the behavior of the cell constituents, especially of mitochondria and plastids, was studied by electron microscopy during the development of the egg and its preceding cells. A degeneration and elimination of mitochondria and plastids was not found in any of the developmental stages. In all growth phases of the archegonium, the plastids may deposit starch which becomes especially frequent in the maturing egg cell. No indications have been observed that new mitochondria or plastids generate from the nuclear evaginations, which often penetrate deeply into the cytoplasm of the maturing and fully developed eggs. A quantitative investigation based on general micrographs elucidates the numerical aspects of the cell constituents during oögenesis. With the increase of cell volume, the numbers of dictyosomes, mitochondria, plastids, and lipid bodies increase. From the stages of the mother cell of the axial row up to that of the mature egg, the cell volume enlarges about 8 times and the nucleus volume about 15 times. Simultaneously, the numbers of mitochondria and plastids increase up to 8 to 15 times. On the basis of these findings, mitochondria and plastids with three-dimensional narrow constrictions are interpreted as divisional stages.

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