scholarly journals High-precision targeting workflow for volume electron microscopy

2021 ◽  
Vol 220 (9) ◽  
Author(s):  
Paolo Ronchi ◽  
Giulia Mizzon ◽  
Pedro Machado ◽  
Edoardo D’Imprima ◽  
Benedikt T. Best ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Single Cells ◽  
Ion Beam ◽  
Mouse Mammary Gland ◽  
Fluorescence Signal ◽  
3D Objects ◽  
Scanning Em ◽  
Automated Data Acquisition ◽  
Volume Electron Microscopy ◽  
Volume Em

Cells are 3D objects. Therefore, volume EM (vEM) is often crucial for correct interpretation of ultrastructural data. Today, scanning EM (SEM) methods such as focused ion beam (FIB)–SEM are frequently used for vEM analyses. While they allow automated data acquisition, precise targeting of volumes of interest within a large sample remains challenging. Here, we provide a workflow to target FIB-SEM acquisition of fluorescently labeled cells or subcellular structures with micrometer precision. The strategy relies on fluorescence preservation during sample preparation and targeted trimming guided by confocal maps of the fluorescence signal in the resin block. Laser branding is used to create landmarks on the block surface to position the FIB-SEM acquisition. Using this method, we acquired volumes of specific single cells within large tissues such as 3D cultures of mouse mammary gland organoids, tracheal terminal cells in Drosophila melanogaster larvae, and ovarian follicular cells in adult Drosophila, discovering ultrastructural details that could not be appreciated before.

scholarly journals FIB-SEM as a Volume Electron Microscopy Approach to Study Cellular Architectures in SARS-CoV-2 and Other Viral Infections: A Practical Primer for a Virologist

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 611
Author(s):  
Valentina Baena ◽  
Ryan Conrad ◽  
Patrick Friday ◽  
Ella Fitzgerald ◽  
Taeeun Kim ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Focused Ion Beam ◽  
Viral Infections ◽  
Ion Beam ◽  
In Vitro Study ◽  
Wide Range ◽  
Sem Imaging ◽  
Volume Electron Microscopy ◽  
Volume Em

The visualization of cellular ultrastructure over a wide range of volumes is becoming possible by increasingly powerful techniques grouped under the rubric “volume electron microscopy” or volume EM (vEM). Focused ion beam scanning electron microscopy (FIB-SEM) occupies a “Goldilocks zone” in vEM: iterative and automated cycles of milling and imaging allow the interrogation of microns-thick specimens in 3-D at resolutions of tens of nanometers or less. This bestows on FIB-SEM the unique ability to aid the accurate and precise study of architectures of virus-cell interactions. Here we give the virologist or cell biologist a primer on FIB-SEM imaging in the context of vEM and discuss practical aspects of a room temperature FIB-SEM experiment. In an in vitro study of SARS-CoV-2 infection, we show that accurate quantitation of viral densities and surface curvatures enabled by FIB-SEM imaging reveals SARS-CoV-2 viruses preferentially located at areas of plasma membrane that have positive mean curvatures.

scholarly journals Fluorescence-based 3D targeting of FIB-SEM acquisition of small volumes in large samples

2021 ◽  
Author(s):  
Paolo Ronchi ◽  
Pedro Machado ◽  
Edoardo D’Imprima ◽  
Giulia Mizzon ◽  
Benedikt T. Best ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Focused Ion Beam ◽  
Single Cells ◽  
Ion Beam ◽  
Three Dimensional ◽  
3D Culture ◽  
Fluorescence Signal ◽  
Three Dimensional Objects ◽  
3D Electron Microscopy ◽  
3D Electron

AbstractCells are three dimensional objects. Therefore, 3D electron microscopy is often crucial for correct interpretation of ultrastructural data. Today samples are frequently imaged in 3D at ultrastructural resolution using volume Scanning Electron Microscopy (SEM) methods such as Focused Ion Beam (FIB) SEM and Serial Block face SEM. While these imaging modalities allow for automated data acquisition, precise targeting of (small) volumes of interest within a large sample remains challenging. Here, we provide an easy and reliable approach to target FIB-SEM acquisition of fluorescently labelled cells or subcellular structures with micrometer precision. The strategy relies on fluorescence preservation during sample preparation and targeting based on confocal acquisition of the fluorescence signal in the resin block. Targeted trimming of the block exposes the cell of interest and laser branding of the surface after trimming creates landmarks to precisely position the FIB-SEM acquisition. Using this method, we acquired volumes of specific single cells within large tissues such as a 3D culture of mouse primary mammary gland organoids, tracheal terminal cells in Drosophila melanogaster larvae and ovarian follicular cells in adult Drosophila, discovering ultrastructural details that could not be appreciated before.SummaryRonchi et al. present a workflow to facilitate the precise targeting of three-dimensional (3D) Electron Microscopy acquisitions, guided by fluorescence. This method allows ultrastructural visualization of single cells within a millimeter-range large specimen, based on molecular identity characterized by fluorescence.

scholarly journals Identifying long-range synaptic inputs using genetically encoded labels and volume electron microscopy

2021 ◽  
Author(s):  
Irene Pilar Ayuso Jimeno ◽  
Paolo Ronchi ◽  
Tianzi Wang ◽  
Catherine Gallori ◽  
Cornelius Thilo Gross
Keyword(s):  
Electron Microscopy ◽  
Long Range ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Anterior Cingulate ◽  
Specific Cell ◽  
Reaction Products ◽  
Synaptic Inputs ◽  
Surfactant Treatment ◽  
Volume Em

Enzymes that facilitate the local deposition of electron dense reaction products have been widely used as labels in electron microscopy (EM). Peroxidases, in particular, can efficiently metabolize 3,3′-diaminobenzidine tetrahydrochloride hydrate (DAB) to produce precipitates with high contrast under EM following heavy metal staining, and can be genetically encoded to facilitate the labeling of specific cell-types or organelles. Nevertheless, the peroxidase/DAB method has so far not been reported to work in combination with 3D volume EM techniques (e.g. Serial blockface electron microscopy, SBEM; Focused ion beam electron microscopy, FIBSEM) because the surfactant treatment needed for efficient reagent penetration disrupts tissue ultrastructure and because these methods require the deposition of large amounts of heavy metals that can obscure DAB precipitates. However, a recently described peroxidase with enhanced enzymatic activity (dAPEX2) appears to successfully deposit EM-visible DAB products in thick tissue without surfactant treatment. Here we demonstrate that multiplexed dAPEX2/DAB tagging is compatible with both FIBSEM and SBEM volume EM approaches and use them to map long-range genetically identified synaptic inputs from the anterior cingulate cortex to the periaqueductal gray in the mouse brain.

Novel cell contact between podocyte microprojections and parietal epithelial cells analyzed by volume electron microscopy

2020 ◽  
Vol 318 (5) ◽  
pp. F1246-F1251
Author(s):  
Christoph Wrede ◽  
Jan Hegermann ◽  
Christian Mühlfeld
Keyword(s):  
Epithelial Cells ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Cell Contact ◽  
Apical Surface ◽  
Electron Microscopic ◽  
Vascular Perfusion ◽  
Scanning Em ◽  
Parietal Epithelial Cells ◽  
Urinary Space

Podocytes are highly specialized cells with a clear cell polarity. It is known that in health and disease, microvilli protrude from the apical surface of the podocytes into the urinary space. As a basis to better understand the podocyte microprojections/microvilli, the present study analyzed their spatial localization, extension, and contact site with parietal epithelial cells (PECs). Using different electron microscopic (EM) techniques, we analyzed renal corpuscles of healthy young adult male C57BL/6 mice fixed by vascular perfusion. Serial block-face scanning EM was used to visualize entire corpuscles, focused ion beam scanning EM was performed to characterize microprojection/microvilli-rich regions at higher magnification, and transmission EM of serial sections was used to analyze the contact zone between podocyte microprojections and PECs. Numerous microprojections originating from the primary processes of podocytes were present in the urinary space in all regions of the corpuscle. They often reached the apical surface of the PEC but did not make junctional contacts. At high resolution, it was observed that the glycocalyx of both cells was in contact. Depending on the distance between podocytes and PECs, these microprojections had a stretched or coiled state. The present study shows that microprojections/microvilli of podocytes are a physiological feature of healthy mouse kidneys and are frequently in contact with the apical surface of PECs, thus spanning the urinary space. It is proposed that podocyte microprojections serve mechanosensory or communicative functions between podocytes and PECs.

scholarly journals Relationship between the Size and Inner Structure of AM Powder Particles

2018 ◽  
Author(s):  
Kateřina Opatová ◽  
Ivana Zetková ◽  
Ludmila Kučerová
Keyword(s):  
Focused Ion Beam ◽  
Complex Geometry ◽  
Phase Distribution ◽  
Ion Beam ◽  
Microscopic Analysis ◽  
Elemental Distribution ◽  
3D Objects ◽  
Manufactured Products ◽  
Metallographic Observation ◽  
Powder Particles

Additive manufacturing (AM) is today’s buzzword—and not only in commercial production. One of the AM techniques produces 3D objects with complex geometry using a laser beam. The relationship between the morphology of individual powder particles and the printing process has not been adequately documented yet. This article presents a detailed microscopic analysis of virgin and reused powder particles of maraging steel. Metallographic observation was performed using a scanning electron microscope (SEM). Detailed analyses of individual particles were carried out using SEM with a focused ion beam (FIB) milling capability. Analyses of elemental distribution and phase distribution were performed using EDS and EBSD, respectively. The findings have led to a better understanding and prediction of defects in additive-manufactured products.

scholarly journals Invaginating Structures in Synapses – Perspective

2021 ◽  
Vol 13 ◽  
Author(s):  
Ronald S. Petralia ◽  
Pamela J. Yao ◽  
Dimitrios Kapogiannis ◽  
Ya-Xian Wang
Keyword(s):  
Electron Microscope ◽  
Light Microscopy ◽  
Muscle Contraction ◽  
Learning And Memory ◽  
Sensory Integration ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Beam Scanning ◽  
Future Studies ◽  
Scanning Em

Invaginating structures are common in the synapses of most animals. However, the details of these invaginating structures remain understudied in part because they are not well resolved in light microscopy and were often misidentified in early electron microscope (EM) studies. Utilizing experimental techniques along with the latest advances in microscopy, such as focused ion beam-scanning EM (FIB-SEM), evidence is gradually building to suggest that the synaptic invaginating structures contribute to synapse development, maintenance, and plasticity. These invaginating structures are most elaborate in synapses mediating rapid integration of signals, such as muscle contraction, mechanoreception, and vision. Here we argue that the synaptic invaginations should be considered in future studies seeking to understand their role in sensory integration and coordination, learning, and memory. We review the various types of invaginating structures in the synapses and discuss their potential functions. We also present several new examples of invaginating structures from a variety of animals including Drosophila and mice, mainly using FIB-SEM, with which we trace the form and arrangement of these structures.

scholarly journals From Light Microscopy to Analytical Scanning Electron Microscopy (SEM) and Focused Ion Beam (FIB)/SEM in Biology: Fixed Coordinates, Flat Embedding, Absolute References

2018 ◽  
Vol 24 (5) ◽  
pp. 526-544 ◽  
Author(s):  
Manja Luckner ◽  
Gerhard Wanner
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Biological Samples ◽  
Focused Ion Beam ◽  
Single Cells ◽  
Ion Beam ◽  
Target Cells ◽  
Wide Range ◽  
Flat Embedding ◽  
Scanning Electron

AbstractCorrelative light and electron microscopy (CLEM) has been in use for several years, however it has remained a costly method with difficult sample preparation. Here, we report a series of technical improvements developed for precise and cost-effective correlative light and scanning electron microscopy (SEM) and focused ion beam (FIB)/SEM microscopy of single cells, as well as large tissue sections. Customized coordinate systems for both slides and coverslips were established for thin and ultra-thin embedding of a wide range of biological specimens. Immobilization of biological samples was examined with a variety of adhesives. For histological sections, a filter system for flat embedding was developed. We validated ultra-thin embedding on laser marked slides for efficient, high-resolution CLEM. Target cells can be re-located within minutes in SEM without protracted searching and correlative investigations were reduced to a minimum of preparation steps, while still reaching highest resolution. The FIB/SEM milling procedure is facilitated and significantly accelerated as: (i) milling a ramp becomes needless, (ii) significant re-deposition of milled material does not occur; and (iii) charging effects are markedly reduced. By optimizing all technical parameters FIB/SEM stacks with 2 nm iso-voxels were achieved over thousands of sections, in a wide range of biological samples.

scholarly journals Identifying Long-range Synaptic Inputs Using Genetically Encoded Labels and Volume Electron Microscopy

2022 ◽  
Author(s):  
Irene P. Ayuso-Jimeno ◽  
Paolo Ronchi ◽  
Tianzi Wang ◽  
Catherine Gallori ◽  
Cornelius T. Gross
Keyword(s):  
Electron Microscopy ◽  
Long Range ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Anterior Cingulate ◽  
Specific Cell ◽  
Reaction Products ◽  
Synaptic Inputs ◽  
Surfactant Treatment ◽  
Volume Em

Abstract Enzymes that facilitate the local deposition of electron dense reaction products have been widely used as labels in electron microscopy (EM). Peroxidases, in particular, can efficiently metabolize 3,3′-diaminobenzidine tetrahydrochloride hydrate (DAB) to produce precipitates with high contrast under EM following heavy metal staining, and can be genetically encoded to facilitate the labeling of specific cell-types or organelles. Nevertheless, the peroxidase/DAB method has so far not been reported to work in combination with 3D volume EM techniques (e.g. Serial blockface electron microscopy, SBEM; Focused ion beam electron microscopy, FIBSEM) because the surfactant treatment needed for efficient reagent penetration disrupts tissue ultrastructure and because these methods require the deposition of large amounts of heavy metals that can obscure DAB precipitates. However, a recently described peroxidase with enhanced enzymatic activity (dAPEX2) appears to successfully deposit EM-visible DAB products in thick tissue without surfactant treatment. Here we demonstrate that multiplexed dAPEX2/DAB tagging is compatible with both FIBSEM and SBEM volume EM approaches and use them to map long-range genetically identified synaptic inputs from the anterior cingulate cortex to the periaqueductal gray in the mouse brain.

A Focused Ion Beam Technique to Electrically Contact the Deep Trench Capacitor of a Single Active Memory Cell in the Sub 0.25µm Technology Regime

2000 ◽  
Author(s):  
Gunnar Zimmermann ◽  
Mark Johnston ◽  
Rolf Christ
Keyword(s):  
Focused Ion Beam ◽  
New Technologies ◽  
Single Cells ◽  
Ion Beam ◽  
Electrical Contact ◽  
Memory Cell ◽  
Beam Current ◽  
Electrical Measurements ◽  
Deep Trench ◽  
Active Memory

Abstract Focused ion beam (FIB) techniques are continuously improved to meet the demands of shrinking device dimensions and new technologies. We developed a simultaneous milling and deposition FIB technique to provide electrical contact to small buried targets in semiconductors. This method is applied to directly connect the deep trench (DT) capacitor of a DRAM single cell in deep submicron technology. By carefully adjusting the deposition parameters (scanned area < (0.3 µm)2, beam current < 20 pA) we are able to influence diameter, depth and Pt fill properties of the hole to meet the very restricted requirements for successful DT connection (hole diameter < 200 nm at DT level). Electrical measurements are performed on DRAM single cells after connecting buried plate (n-band), p-well, wordline, bitline and DT. The probe pads were Pt, deposited with ion beam assistance, on top of highly insulating SiOx, deposited with electron beam assistance by using a dualbeam FIB. The read and write conditions of an active memory cell are studied. The presented method increases the capabilities to localize and characterize trench related failure mechanisms.

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