scholarly journals Three-dimensional imaging of podocyte ultrastructure using FE-SEM and FIB-SEM tomography

2019 ◽  
Vol 379 (2) ◽  
pp. 245-254 ◽  
Author(s):  
Takayuki Miyaki ◽  
Yuto Kawasaki ◽  
Yasue Hosoyamada ◽  
Takashi Amari ◽  
Mui Kinoshita ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
3D Visualization ◽  
Ion Beam ◽  
Three Dimensional ◽  
3D Analysis ◽  
Subcellular Compartment ◽  
Advantages And Disadvantages ◽  
Powerful Approach ◽  
Foot Process ◽  
Serial Images

AbstractPodocytes are specialized epithelial cells used for glomerular filtration in the kidney. They can be divided into the cell body, primary process and foot process. Here, we describe two useful methods for the three-dimensional(3D) visualization of these subcellular compartments in rodent podocytes. The first method, field-emission scanning electron microscopy (FE-SEM) with conductive staining, is used to visualize the luminal surface of numerous podocytes simultaneously. The second method, focused-ion beam SEM (FIB-SEM) tomography, allows the user to obtain serial images from different depths of field, or Z-stacks, of the glomerulus. This allows for the 3D reconstruction of podocyte ultrastructure, which can be viewed from all angles, from a single image set. This is not possible with conventional FE-SEM. The different advantages and disadvantages of FE-SEM and FIB-SEM tomography compensate for the weaknesses of the other. The combination renders a powerful approach for the 3D analysis of podocyte ultrastructure. As a result, we were able to identify a new subcellular compartment of podocytes, “ridge-like prominences” (RLPs).

scholarly journals Multi-Scale 3D Cryo-Correlative Microscopy for Vitrified Cells

2020 ◽  
Author(s):  
Gong-Her Wu ◽  
Patrick G. Mitchell ◽  
Jesus G. Galaz-Montoya ◽  
Corey W. Hecksel ◽  
Emily M. Sontag ◽  
...  
Keyword(s):  
Large Scale ◽  
Focused Ion Beam ◽  
Electron Tomography ◽  
3D Visualization ◽  
Ion Beam ◽  
Three Dimensional ◽  
Yeast Cells ◽  
Multi Scale ◽  
Fluorescence Confocal Microscopy ◽  
Cryo Electron Tomography

SUMMARYThree-dimensional (3D) visualization of vitrified cells can uncover structures of subcellular complexes without chemical fixation or staining. Here, we present a pipeline integrating three imaging modalities to visualize the same specimen at cryogenic temperature at different scales: cryo-fluorescence confocal microscopy, volume cryo-focused ion beam scanning electron microscopy, and transmission cryo-electron tomography. Our proof-of-concept benchmark revealed the 3D distribution of organelles and subcellular structures in whole heat-shocked yeast cells, including the ultrastructure of protein inclusions that recruit fluorescently-labelled chaperone Hsp104. Since our workflow efficiently integrates imaging at three different scales and can be applied to other types of cells, it could be used for large-scale phenotypic studies of frozen-hydrated specimens in a variety of healthy and diseased conditions with and without treatments.

scholarly journals 3D Ultrastructural Study of Synapses in the Human Entorhinal Cortex

2020 ◽  
Vol 31 (1) ◽  
pp. 410-425 ◽  
Author(s):  
M Domínguez-Álvaro ◽  
M Montero-Crespo ◽  
L Blazquez-Llorca ◽  
J DeFelipe ◽  
L Alonso-Nanclares
Keyword(s):  
Entorhinal Cortex ◽  
Focused Ion Beam ◽  
Functional Organization ◽  
Structural Parameters ◽  
Ion Beam ◽  
Three Dimensional ◽  
Ultrastructural Level ◽  
3D Analysis ◽  
Synaptic Organization ◽  
Health And Disease

Abstract The entorhinal cortex (EC) is a brain region that has been shown to be essential for memory functions and spatial navigation. However, detailed three-dimensional (3D) synaptic morphology analysis and identification of postsynaptic targets at the ultrastructural level have not been performed before in the human EC. In the present study, we used Focused Ion Beam/Scanning Electron Microscopy to perform a 3D analysis of the synapses in the neuropil of medial EC in layers II and III from human brain autopsies. Specifically, we studied synaptic structural parameters of 3561 synapses, which were fully reconstructed in 3D. We analyzed the synaptic density, 3D spatial distribution, and type (excitatory and inhibitory), as well as the shape and size of each synaptic junction. Moreover, the postsynaptic targets of synapses could be clearly determined. The present work constitutes a detailed description of the synaptic organization of the human EC, which is a necessary step to better understand the functional organization of this region in both health and disease.

Three Dimensional (3D) Visualization of Damage in Metal-Ceramic Nanolayers by Focused Ion Beam (FIB) Serial Sectioning

2008 ◽  
Vol 14 (S2) ◽  
pp. 140-141 ◽  
Author(s):  
N Chawla ◽  
DRP Singh
Keyword(s):  
New Mexico ◽  
Focused Ion Beam ◽  
3D Visualization ◽  
Ion Beam ◽  
Three Dimensional ◽  
Serial Sectioning ◽  
Metal Ceramic

Extended abstract of a paper presented at Microscopy and Microanalysis 2008 in Albuquerque, New Mexico, USA, August 3 – August 7, 2008

3D Observation of Elemental Distribution of Si-Device using a Dedicated FIB/STEM System

2005 ◽  
Author(s):  
T. Yaguchi ◽  
M. Konno ◽  
T. Kamino ◽  
M. Ogasawara ◽  
K. Kaji ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Ion Beam ◽  
Three Dimensional ◽  
Elemental Distribution ◽  
Multivariate Statistical ◽  
X Ray ◽  
Sample Rotation ◽  
Transmission Electron ◽  
Scanning Transmission ◽  
Elemental Maps

Abstract A technique for preparation of a pillar shaped sample and its multi-directional observation of the sample using a focused ion beam (FIB) / scanning transmission electron microscopy (STEM) system has been developed. The system employs an FIB/STEM compatible sample rotation holder with a specially designed rotation mechanism, which allows the sample to be rotated 360 degrees [1-3]. This technique was used for the three dimensional (3D) elemental mapping of a contact plug of a Si device in 90 nm technology. A specimen containing a contact plug was shaped to a pillar sample with a cross section of 200 nm x 200 nm and a 5 um length. Elemental analysis was performed with a 200 kV HD-2300 STEM equipped with the EDAX genesis Energy dispersive X-ray spectroscopy (EDX) system. Spectrum imaging combined with multivariate statistical analysis (MSA) [4, 5] was used to enhance the weak X-ray signals of the doped area, which contain a low concentration of As-K. The distributions of elements, especially the dopant As, were successfully enhanced by MSA. The elemental maps were .. reconstructed from the maps.

scholarly journals Investigation of the selective color-changing mechanism of Dynastes tityus beetle (Coleoptera: Scarabaeidae)

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jiyu Sun ◽  
Wei Wu ◽  
Limei Tian ◽  
Wei Li ◽  
Fang Zhang ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Color Change ◽  
Incidence Angle ◽  
Ion Beam ◽  
Three Dimensional ◽  
Water Infiltration ◽  
Photonic Materials ◽  
Local Color ◽  
Water Contact ◽  
Color Changes

AbstractNot only does the Dynastes tityus beetle display a reversible color change controlled by differences in humidity, but also, the elytron scale can change color from yellow-green to deep-brown in specified shapes. The results obtained by focused ion beam-scanning electron microscopy (FIB-SEM), show that the epicuticle (EPI) is a permeable layer, and the exocuticle (EXO) is a three-dimensional photonic crystal. To investigate the mechanism of the reversible color change, experiments were conducted to determine the water contact angle, surface chemical composition, and optical reflectance, and the reflective spectrum was simulated. The water on the surface began to permeate into the elytron via the surface elemental composition and channels in the EPI. A structural unit (SU) in the EXO allows local color changes in varied shapes. The reflectance of both yellow-green and deep-brown elytra increases as the incidence angle increases from 0° to 60°. The microstructure and changes in the refractive index are the main factors that influence the process of reversible color change. According to the simulation, the lower reflectance causing the color change to deep-brown results from water infiltration, which increases light absorption. Meanwhile, the waxy layer has no effect on the reflection of light. This study lays the foundation to manufacture engineered photonic materials that undergo controllable changes in iridescent color.

scholarly journals Protocol for preparation of heterogeneous biological samples for 3D electron microscopy: a case study for insects

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alexey A. Polilov ◽  
Anastasia A. Makarova ◽  
Song Pang ◽  
C. Shan Xu ◽  
Harald Hess
Keyword(s):  
Electron Microscopy ◽  
Biological Samples ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Three Dimensional ◽  
Entire Volume ◽  
Simple Protocol ◽  
3D Electron Microscopy ◽  
3D Em

AbstractModern morphological and structural studies are coming to a new level by incorporating the latest methods of three-dimensional electron microscopy (3D-EM). One of the key problems for the wide usage of these methods is posed by difficulties with sample preparation, since the methods work poorly with heterogeneous (consisting of tissues different in structure and in chemical composition) samples and require expensive equipment and usually much time. We have developed a simple protocol allows preparing heterogeneous biological samples suitable for 3D-EM in a laboratory that has a standard supply of equipment and reagents for electron microscopy. This protocol, combined with focused ion-beam scanning electron microscopy, makes it possible to study 3D ultrastructure of complex biological samples, e.g., whole insect heads, over their entire volume at the cellular and subcellular levels. The protocol provides new opportunities for many areas of study, including connectomics.

scholarly journals Site Specific Three-dimensional Structural Analysis in Tissues and Cells Using Automated DualBeam Slice &View

2007 ◽  
Vol 15 (2) ◽  
pp. 26-31 ◽  
Author(s):  
Ben Lich
Keyword(s):  
Electron Microscopy ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Three Dimensional ◽  
Surface Region ◽  
Dimensional Structure ◽  
Near Surface ◽  
Imaging Capability ◽  
Fluorescent Markers ◽  
Confocal And Electron Microscopy

DualBeam instruments that combine the imaging capability of scanning electron microscopy (SEM) with the cutting and deposition capability of a focused ion beam (FIB) provide biologists with a powerful tool for investigating three-dimensional structure with nanoscale (1 nm-100 nm) resolution. Ever since Van Leeuwenhoek used the first microscope to describe bacteria more than 300 years ago, microscopy has played a central role in scientists' efforts to understand biological systems. Light microscopy is generally limited to a useful resolution of about a micrometer. More recently the use of confocal and electron microscopy has enabled investigations at higher resolution. Used with fluorescent markers, confocal microscopy can detect and localize molecular scale features, but its imaging resolution is still limited. SEM is capable of nanometer resolution, but is limited to the near surface region of the sample.

Molding of Three-Dimensional Microcomponents

2006 ◽  
Author(s):  
W. N. P. Hung ◽  
M. M. Agnihotri ◽  
M. Y. Ali ◽  
S. Yuan
Keyword(s):  
Focused Ion Beam ◽  
New Technologies ◽  
Electrical Discharge Machining ◽  
Ion Beam ◽  
Semiconductor Industry ◽  
Three Dimensional ◽  
Cost Effective ◽  
Molecular Structures ◽  
Minimum Size ◽  
Ion Beam Sputtering

Traditional micromanufacturing has been developed for semiconductor industry. Selected micro electrical mechanical systems (MEMS) have been successfully developed and implemented in industry. Since current MEMS are designed for manufacture using microelectronics processes, they are limited to two-dimensional profiles and semiconductor based materials. Such shape and material constraints would exclude many applications that require biocompatibility, dynamic stress, and high ductility. New technologies are sought to fabricate three dimensional microcomponents using robust materials for demanding applications. To be cost effective, such microdevices must be economically mass producible. Molding is one of the promising replication techniques to mass produce components from polymers and polymer-based composites. This paper presents the development of a micromolding process to produce thermoplastic microcomponents. Mold design required precision fitting and was integrated with a vacuum pump to minimize air trap in mold cavities. Nickel and aluminum mold inserts were used for the study; their cavities were fabricated by combinations of available micromachining processes like laser micromachining, micromilling, micro electrical discharge machining, and focused ion beam sputtering. High and low density polyethylene, polystyrene polymers were used for this study. The effects of polymer molecular structures, molding temperature, time, and pressure on molding results were studied. Simulation of stress in the microcomponents, plastic flow in microchannels, and mold defects was performed and compare with experimental data. The research results showed that a microcomponent can be fabricated to the minimum size of 10 ± 1μm (0.0004 inch) with surface roughness <10 nm Rt. Molding of micro-size geartrains and orthopedic meso-size fasteners was completed to illustrate the capability of this process.

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