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 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 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.

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 Quantitative method for estimating stain density in electron microscopy of conventionally prepared biological specimens

2019 ◽  
Author(s):  
Andrea Fera ◽  
Qianping He ◽  
Guofeng Zhang ◽  
Richard D. Leapman
Keyword(s):  
Electron Microscopy ◽  
Focused Ion Beam ◽  
Electron Tomography ◽  
Blood Platelets ◽  
Ion Beam ◽  
Simple Expression ◽  
Heavy Atoms ◽  
3D Electron Microscopy ◽  
Block Face ◽  
Microscopy Techniques

SummaryStain density is an important parameter for optimizing the quality of ultrastructural data obtained from several types of 3D electron microscopy techniques, including serial block-face electron microscopy (SBEM), and focused ion beam scanning electron microscopy (FIB-SEM). Here, we show how some straightforward measurements in the TEM can be used to determine the stain density based on a simple expression that we derive. Numbers of stain atoms per unit volume are determined from the measured ratio of the bright-field intensities from regions of the specimen that contain both pure embedding material and the embedded biological structures of interest. The determination only requires knowledge of the section thickness, which can either be estimated from the microtome setting, or from low-dose electron tomography, and the elastic scattering cross section for the heavy atoms used to stain the specimen. The method is tested on specimens of embedded blood platelets, brain tissue, and liver tissue.

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.

Structuring of Conducting Polymers by Ion Implantation

1995 ◽  
Vol 396 ◽  
Author(s):  
Klaus Edinger ◽  
Stefanie Schiestel ◽  
Gerhard K. Wolf
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Ion Implantation ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Electrochemical Behaviour ◽  
Three Dimensional ◽  
Electrochemical Treatment ◽  
Lateral Structures ◽  
Scanning Electron

AbstractConducting polypyrrole polymer films have been modified by ion implantation. The resulting cross linking leads to changes in resistivity and electrochemical behaviour. By ion implantation through masks or with a focused ion beam lateral structures can be produced which can be imaged by scanning electron microscopy and optical absorption. The implanted polypyrrole layers can be removed by electrochemical treatment while not implanted regions can be electroplated. Therefore in combination with electrochemical treatment three dimensional structures have been generated and were investigated by atomic force microscopy. In order to study structures in the submicrometer range implantation experiments with a focused ion beam were performed and the minimal line widths were investigated by scanning electron microscopy.

scholarly journals Tb3+-doped LaF3nanocrystals for correlative cathodoluminescence electron microscopy imaging with nanometric resolution in focused ion beam-sectioned biological samples

Nanoscale ◽  
2017 ◽  
Vol 9 (13) ◽  
pp. 4383-4387 ◽  
Author(s):  
K. Keevend ◽  
M. Stiefel ◽  
A. L. Neuer ◽  
M. T. Matter ◽  
A. Neels ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Biological Samples ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Microscopy Imaging
Sign in / Sign up

Export Citation Format

Share Document