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 ◽  
Bovine Serum Albumin ◽  
Fluorescence Microscopy ◽  
Light And Electron Microscopy ◽  
Three Dimensional ◽  
Fiducial Marker ◽  
Regions Of Interest ◽  
Dynamic Information ◽  
Endosomal System ◽  
2D And 3D

Correlative light and electron microscopy (CLEM) can infer molecular, functional and dynamic information to ultrastructure by linking information of different imaging modalities. One of the main challenges, especially in 3D-CLEM, is the accurate registration of fluorescent signals to electron microscopy (EM). Here, we present fluorescent BSA-gold (fBSA-Au), a bimodal endocytic tracer as fiducial marker for 2D and 3D CLEM applications. fBSA-Au consists of colloidal gold (Au) particles stabilized with fluorescent bovine serum albumin (BSA). The conjugate is efficiently endocytosed and distributed throughout the 3D endo-lysosomal network of the cells, and has an excellent visibility both in fluorescence microscopy (FM) and EM. We demonstrate the use of fBSA-Au in several 2D and 3D CLEM applications using Tokuyasu cryosections, resin-embedded material, and cryo-EM. As a fiducial marker, fBSA-Au facilitates rapid registration of regions of interest between FM and EM modalities and enables accurate (50-150 nm) correlation of fluorescence to EM data. Endocytosed fBSA-Au benefits from a homogenous 3D distribution throughout the endosomal system within the cell, and does not obscure any cellular ultrastructure. The broad applicability and visibility in both modalities makes fBSA-Au an excellent endocytic fiducial marker for 2D and 3D (cryo-)CLEM applications.

Quantitative correlative microscopy reveals the ultrastructural distribution of endogenous endosomal proteins

2021 ◽  
Vol 221 (1) ◽  
Author(s):  
Jan van der Beek ◽  
Cecilia de Heus ◽  
Nalan Liv ◽  
Judith Klumperman
Keyword(s):  
Electron Microscopy ◽  
Fluorescence Microscopy ◽  
Functional Organization ◽  
Light And Electron Microscopy ◽  
Correlative Microscopy ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Endocytic Vesicles ◽  
Endosomal System

The key endosomal regulators Rab5, EEA1, and APPL1 are frequently applied in fluorescence microscopy to mark early endosomes, whereas Rab7 is used as a marker for late endosomes and lysosomes. However, endogenous levels of these proteins localize poorly in immuno-EM, and systematic studies on their native ultrastructural distributions are lacking. To address this gap, we here present a quantitative, on-section correlative light and electron microscopy (CLEM) approach. Using the sensitivity of fluorescence microscopy, we label hundreds of organelles that are subsequently visualized by EM and classified by ultrastructure. We show that Rab5 predominantly marks small, endocytic vesicles and early endosomes. EEA1 colocalizes with Rab5 on early endosomes, but unexpectedly also labels Rab5-negative late endosomes, which are positive for PI(3)P but lack Rab7. APPL1 is restricted to small Rab5-positive, tubulo-vesicular profiles. Rab7 primarily labels late endosomes and lysosomes. These data increase our understanding of the structural–functional organization of the endosomal system and introduce quantitative CLEM as a sensitive alternative for immuno-EM.

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 The adult oligodendrocyte can participate in remyelination

2018 ◽  
Vol 115 (50) ◽  
pp. E11807-E11816 ◽  
Author(s):  
Ian D. Duncan ◽  
Abigail B. Radcliff ◽  
Moones Heidari ◽  
Grahame Kidd ◽  
Benjamin K. August ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Multiple Sclerosis ◽  
Nonhuman Primates ◽  
Light And Electron Microscopy ◽  
Vitamin B ◽  
Oligodendrocyte Progenitor Cells ◽  
Large Animal ◽  
Myelin Sheaths ◽  
Large Animal Models ◽  
2D And 3D

Endogenous remyelination of the CNS can be robust and restore function, yet in multiple sclerosis it becomes less complete with time. Promoting remyelination is a major therapeutic goal, both to restore function and to protect axons from degeneration. Remyelination is thought to depend on oligodendrocyte progenitor cells, giving rise to nascent remyelinating oligodendrocytes. Surviving, mature oligodendrocytes are largely regarded as being uninvolved. We have examined this question using two large animal models. In the first model, there is extensive demyelination and remyelination of the CNS, yet oligodendrocytes survive, and in recovered animals there is a mix of remyelinated axons interspersed between mature, thick myelin sheaths. Using 2D and 3D light and electron microscopy, we show that many oligodendrocytes are connected to mature and remyelinated myelin sheaths, which we conclude are cells that have reextended processes to contact demyelinated axons while maintaining mature myelin internodes. In the second model in vitamin B12-deficient nonhuman primates, we demonstrate that surviving mature oligodendrocytes extend processes and ensheath demyelinated axons. These data indicate that mature oligodendrocytes can participate in remyelination.

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