Faculty Opinions recommendation of Correlated three-dimensional light and electron microscopy reveals transformation of mitochondria during apoptosis.

2007 ◽  
Author(s):  
Richard Youle
Keyword(s):  
Electron Microscopy ◽  
Light And Electron Microscopy ◽  
Three Dimensional

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 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.

Correlated three-dimensional light and electron microscopy reveals transformation of mitochondria during apoptosis

2007 ◽  
Vol 9 (9) ◽  
pp. 1057-1065 ◽  
Author(s):  
Mei G. Sun ◽  
James Williams ◽  
Cristina Munoz-Pinedo ◽  
Guy A. Perkins ◽  
Joshua M. Brown ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Light And Electron Microscopy ◽  
Three Dimensional

scholarly journals A CLEM approach to access to the ultrastructure at the graft interface in Arabidopsis thaliana

2021 ◽  
Author(s):  
Clément Chambaud ◽  
Sarah Jane Cookson ◽  
Nathalie Ollat ◽  
Emmanuelle M. F. Bayer ◽  
Lysiane Brocard
Keyword(s):  
Electron Microscopy ◽  
Arabidopsis Thaliana ◽  
Endoplasmic Reticulum ◽  
Cell Wall ◽  
Light And Electron Microscopy ◽  
Three Dimensional ◽  
Cellular Events ◽  
Membrane Tethering ◽  
Secondary Plasmodesmata ◽  
Graft Interface

Despite recent progress in our understanding of the graft union formation, we still know little about the cellular events underlying the grafting process. This is partially due to the difficulty of reliably targeting the graft interface in electron microscopy to study its ultrastructure and three-dimensional architecture. To overcome this technological bottleneck, we developed a correlative light electron microscopy approach (CLEM) to study the graft interface with high ultrastructural resolution. Grafting hypocotyls of Arabidopsis thaliana lines expressing YFP or mRFP in the endoplasmic reticulum allowed the efficient targeting of the grafting interface for under light and electron microscopy. To explore the potential of our method to study sub-cellular events at the graft interface, we focused on the formation of secondary plasmodesmata (PD) between the grafted partners. We showed that 4 classes of PD were formed at the interface and that PD introgression into the call wall was initiated equally by both partners. Moreover, the success of PD formation appeared not systematic with a third of PD not spanning the cell wall entirely. Characterizing the ultrastructural characteristics of these failed PD gives us insights into the process of secondary PD biogenesis. We showed that the thinning of the cell wall and the endoplasmic reticulum-plasma membrane tethering seem to be required for the establishment of symplastic connections between the scion and the rootstock. The resolution reached in this work shows that our CLEM method offer a new scale to the study for biological processes requiring the combination of light and electron microscopy.

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.

Ultrastructure et représentation spatiale du manteau fongique des ectomycorhizes

1979 ◽  
Vol 57 (21) ◽  
pp. 2319-2324 ◽  
Author(s):  
D. G. Strullu
Keyword(s):  
Electron Microscopy ◽  
Pseudotsuga Menziesii ◽  
Light And Electron Microscopy ◽  
Three Dimensional ◽  
Douglas Fir ◽  
Dimensional Structure ◽  
Three Dimensional Structure

A study of mycorrhizas of Douglas fir (Pseudotsuga menziesii Mirb.) by light and electron microscopy has allowed us to show the organization of the mantle. A three-dimensional structure of the fungal mantle is proposed. The outer plectenchyma is characterized by free hyphae; these hyphae are devoid of cytoplasmic contents. The inner plectenchyma shows closely interwoven hyphae; the fungal cells have a living aspect and a glycogen-rich cytoplasm.

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 Fluorescence photooxidation with eosin: a method for high resolution immunolocalization and in situ hybridization detection for light and electron microscopy.

1994 ◽  
Vol 126 (4) ◽  
pp. 901-910 ◽  
Author(s):  
T J Deerinck ◽  
M E Martone ◽  
V Lev-Ram ◽  
D P Green ◽  
R Y Tsien ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
In Situ Hybridization ◽  
High Resolution ◽  
Light And Electron Microscopy ◽  
Three Dimensional ◽  
Substantial Improvement ◽  
Electron Microscopic ◽  
Simple Method ◽  
High Voltage Electron Microscopy

A simple method is described for high-resolution light and electron microscopic immunolocalization of proteins in cells and tissues by immunofluorescence and subsequent photooxidation of diaminobenzidine tetrahydrochloride into an insoluble osmiophilic polymer. By using eosin as the fluorescent marker, a substantial improvement in sensitivity is achieved in the photooxidation process over other conventional fluorescent compounds. The technique allows for precise correlative immunolocalization studies on the same sample using fluorescence, transmitted light and electron microscopy. Furthermore, because eosin is smaller in size than other conventional markers, this method results in improved penetration of labeling reagents compared to gold or enzyme based procedures. The improved penetration allows for three-dimensional immunolocalization using high voltage electron microscopy. Fluorescence photooxidation can also be used for high resolution light and electron microscopic localization of specific nucleic acid sequences by in situ hybridization utilizing biotinylated probes followed by an eosin-streptavidin conjugate.

scholarly journals Bimodal Endocytic Probe for Three-Dimensional Correlative Light and Electron Microscopy

2021 ◽  
Author(s):  
Job Fermie ◽  
Leanne de Jager ◽  
Helen Foster ◽  
Tineke Veenendaal ◽  
Cecilia de Heus ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Light And Electron Microscopy ◽  
Three Dimensional
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