scholarly journals Correlation of organelle dynamics between light microscopic live imaging and electron microscopic 3D architecture using FIB-SEM

Microscopy ◽  
2020 ◽  
Author(s):  
Keisuke Ohta ◽  
Shingo Hirashima ◽  
Yoshihiro Miyazono ◽  
Akinobu Togo ◽  
Kei-ichiro Nakamura
Keyword(s):  
Electron Microscopy ◽  
Cell Biology ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Light And Electron Microscopy ◽  
Three Dimensional ◽  
Optical Microscope ◽  
Live Imaging ◽  
Reconstruction Technique ◽  
Electron Microscopic

Abstract Correlative light and electron microscopy (CLEM) methods combined with live imaging can be applied to understand the dynamics of organelles. Although recent advances in cell biology and light microscopy have helped in visualizing the details of organelle activities, observing their ultrastructure or organization of surrounding microenvironments is a challenging task. Therefore, CLEM, which allows us to observe the same area as an optical microscope with an electron microscope, has become a key technique in cell biology. Unfortunately, most CLEM methods have technical drawbacks, and many researchers face difficulties in applying CLEM methods. Here, we propose a live three-dimensional CLEM method, combined with a three-dimensional reconstruction technique using focused ion beam scanning electron microscopy tomography, as a solution to such technical barriers. We review our method, the associated technical limitations and the options considered to perform live CLEM.

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 Volume electron microscopy: analyzing the lung

2020 ◽  
Author(s):  
Jan Philipp Schneider ◽  
Jan Hegermann ◽  
Christoph Wrede
Keyword(s):  
Electron Microscopy ◽  
Focused Ion Beam ◽  
Electron Tomography ◽  
Ion Beam ◽  
Three Dimensional ◽  
Alveolar Epithelium ◽  
Practical Implementation ◽  
Electron Microscopic ◽  
Array Tomography ◽  
Three Dimensional Models

AbstractSince its entry into biomedical research in the first half of the twentieth century, electron microscopy has been a valuable tool for lung researchers to explore the lung’s delicate ultrastructure. Among others, it proved the existence of a continuous alveolar epithelium and demonstrated the surfactant lining layer. With the establishment of serial sectioning transmission electron microscopy, as the first “volume electron microscopic” technique, electron microscopy entered the third dimension and investigations of the lung’s three-dimensional ultrastructure became possible. Over the years, further techniques, ranging from electron tomography over serial block-face and focused ion beam scanning electron microscopy to array tomography became available. All techniques cover different volumes and resolutions, and, thus, different scientific questions. This review gives an overview of these techniques and their application in lung research, focusing on their fields of application and practical implementation. Furthermore, an introduction is given how the output raw data are processed and the final three-dimensional models can be generated.

scholarly journals In situ Microfluidic Cryofixation for Cryo Focused Ion Beam Milling and Cryo Electron Tomography

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Marie Fuest ◽  
Miroslava Schaffer ◽  
Giovanni Marco Nocera ◽  
Rodrigo I. Galilea-Kleinsteuber ◽  
Jan-Erik Messling ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Light Microscope ◽  
Focused Ion Beam ◽  
Electron Tomography ◽  
Ion Beam ◽  
Light And Electron Microscopy ◽  
Live Imaging ◽  
Cryo Electron Tomography

AbstractWe present a microfluidic platform for studying structure-function relationships at the cellular level by connecting video rate live cell imaging with in situ microfluidic cryofixation and cryo-electron tomography of near natively preserved, unstained specimens. Correlative light and electron microscopy (CLEM) has been limited by the time required to transfer live cells from the light microscope to dedicated cryofixation instruments, such as a plunge freezer or high-pressure freezer. We recently demonstrated a microfluidic based approach that enables sample cryofixation directly in the light microscope with millisecond time resolution, a speed improvement of up to three orders of magnitude. Here we show that this cryofixation method can be combined with cryo-electron tomography (cryo-ET) by using Focused Ion Beam milling at cryogenic temperatures (cryo-FIB) to prepare frozen hydrated electron transparent sections. To make cryo-FIB sectioning of rapidly frozen microfluidic channels achievable, we developed a sacrificial layer technique to fabricate microfluidic devices with a PDMS bottom wall <5 µm thick. We demonstrate the complete workflow by rapidly cryo-freezing Caenorhabditis elegans roundworms L1 larvae during live imaging in the light microscope, followed by cryo-FIB milling and lift out to produce thin, electron transparent sections for cryo-ET imaging. Cryo-ET analysis of initial results show that the structural preservation of the cryofixed C. elegans was suitable for high resolution cryo-ET work. The combination of cryofixation during live imaging enabled by microfluidic cryofixation with the molecular resolution capabilities of cryo-ET offers an exciting avenue to further advance space-time correlative light and electron microscopy (st-CLEM) for investigation of biological processes at high resolution in four dimensions.

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.

FIB-SEM: An Additional Technique for Investigating Internal Structure of Pollen Walls

2013 ◽  
Vol 19 (6) ◽  
pp. 1535-1541 ◽  
Author(s):  
Alisoun House ◽  
Kevin Balkwill
Keyword(s):  
Electron Microscopy ◽  
Internal Structure ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Three Dimensional ◽  
Pollen Grains ◽  
Grain Morphology ◽  
Pollen Grain ◽  
Pollen Wall ◽  
Scanning Electron

AbstractPollen grain morphology has been widely used in the classification of the Acanthaceae, where external pollen wall features have proved useful in determining relationships between taxa. Although detailed information has been accumulated using light microscopy, transmission electron microscopy and scanning electron microscopy (SEM) techniques, internal pollen wall features lack investigation and the techniques are cumbersome. A new technique involving precise cross sectioning or slicing of pollen grains at a selected position for examining wall ultrastructure, using a focused ion beam-scanning electron microscope (FIB-SEM), has been explored and promising results have been obtained. The FIB-SEM offers a good technique for reliable, high resolution, three-dimensional (3D) viewing of the internal structure of the pollen grain wall.

Coupled Mechanical and Electrochemical Analyses of Three-Dimensional Reconstructed LiFePO4 by Focused Ion Beam/Scanning Electron Microscopy in Lithium-Ion Batteries

2018 ◽  
Vol 16 (1) ◽  
Author(s):  
Sangwook Kim ◽  
Hongjiang Chen ◽  
Hsiao-Ying Shadow Huang
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Lithium Ion Batteries ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Three Dimensional ◽  
Lithium Ion ◽  
Electrochemical Degradation ◽  
Total Polarization ◽  
Scanning Electron

Limited lifetime and performance degradation in lithium ion batteries in electrical vehicles and power tools is still a challenging obstacle which results from various interrelated processes, especially under specific conditions such as higher discharging rates (C-rates) and longer cycles. To elucidate these problems, it is very important to analyze electrochemical degradation from a mechanical stress point of view. Specifically, the goal of this study is to investigate diffusion-induced stresses and electrochemical degradation in three-dimensional (3D) reconstructed LiFePO4. We generate a reconstructed microstructure by using a stack of focused ion beam-scanning electron microscopy (FIB/SEM) images combined with an electrolyte domain. Our previous two-dimensional (2D) finite element model is further improved to a 3D multiphysics one, which incorporates both electrochemical and mechanical analyses. From our electrochemistry model, we observe 95.6% and 88.3% capacity fade at 1.2 C and 2 C, respectively. To investigate this electrochemical degradation, we present concentration distributions and von Mises stress distributions across the cathode with respect to the depth of discharge (DoD). Moreover, electrochemical degradation factors such as total polarization and over-potential are also investigated under different C-rates. Further, higher total polarization is observed at the end of discharging, as well as at the early stage of discharging. It is also confirmed that lithium intercalation at the electrode-electrolyte interface causes higher over-potential at specific DoDs. At the region near the separator, a higher concentration gradient and over-potential are observed. We note that higher over-potential occurs on the surface of electrode, and the resulting concentration gradient and mechanical stresses are observed in the same regions. Furthermore, mechanical stress variations under different C-rates are quantified during the discharging process. With these coupled mechanical and electrochemical analyses, the results of this study may be helpful for detecting particle crack initiation.

Sign in / Sign up

Export Citation Format

Share Document