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.

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 From tissue retrieval to electron tomography: nanoscale characterization of the interface between bone and bioactive glass

2021 ◽  
Vol 18 (182) ◽  
pp. 20210181
Author(s):  
Chiara Micheletti ◽  
Pedro Henrique Silva Gomes-Ferreira ◽  
Travis Casagrande ◽  
Paulo Noronha Lisboa-Filho ◽  
Roberta Okamoto ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Bioactive Glass ◽  
Focused Ion Beam ◽  
Electron Tomography ◽  
Ion Beam ◽  
Three Dimensions ◽  
Transmission Electron Microscopy Analysis ◽  
Dental Procedures ◽  
Transmission Electron

The success of biomaterials for bone regeneration relies on many factors, among which osseointegration plays a key role. Biogran (BG) is a bioactive glass commonly employed as a bone graft in dental procedures. Despite its use in clinical practice, the capability of BG to promote osseointegration has never been resolved at the nanoscale. In this paper, we present the workflow for characterizing the interface between newly formed bone and BG in a preclinical rat model. Areas of bone–BG contact were first identified by backscattered electron imaging in a scanning electron microscope. A focused ion beam in situ lift-out protocol was employed to prepare ultrathin samples for transmission electron microscopy analysis. The bone–BG gradual interface, i.e. the biointerphase, was visualized at the nanoscale with unprecedented resolution thanks to scanning transmission electron microscopy. Finally, we present a method to view the bone–BG interface in three dimensions using electron tomography.

scholarly journals In-cell quantitative structural imaging of phytoplankton using 3D electron microscopy

2020 ◽  
Author(s):  
Clarisse Uwizeye ◽  
Johan Decelle ◽  
Pierre-Henri Jouneau ◽  
Benoit Gallet ◽  
Jean-Baptiste Keck ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Biology ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Phytoplankton Diversity ◽  
Cellular Energy ◽  
Unicellular Eukaryotes ◽  
3D Electron Microscopy ◽  
Abiotic And Biotic Factors ◽  
A Minor

AbstractPhytoplankton is a minor fraction of the global biomass playing a major role in primary production and climate. Despite improved understanding of phytoplankton diversity and genomics, we lack nanoscale subcellular imaging approaches to understand their physiology and cell biology. Here, we present a complete Focused Ion Beam - Scanning Electron Microscopy (FIB-SEM) workflow (from sample preparation to image processing) to generate nanometric 3D phytoplankton models. Tomograms of entire cells, representatives of six ecologically-successful phytoplankton unicellular eukaryotes, were used for quantitative morphometric analysis. Besides lineage-specific cellular architectures, we observed common features related to cellular energy management: i) conserved cell-volume fractions occupied by the different organelles; ii) consistent plastid-mitochondria interactions, iii) constant volumetric ratios in these energy-producing organelles. We revealed detailed subcellular features related to chromatin organization and to biomineralization. Overall, this approach opens new perspectives to study phytoplankton acclimation responses to abiotic and biotic factors at a relevant biological scale.

scholarly journals Targeted Studies Using Serial Block Face and Focused Ion Beam Scan Electron Microscopy

10.3791/59480 ◽  
2019 ◽  
Author(s):  
Christopher J. Guérin ◽  
Anna Kremer ◽  
Peter Borghgraef ◽  
Saskia Lippens
Keyword(s):  
Electron Microscopy ◽  
Scan Electron Microscopy ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Block Face

New Techniques for the Nanostructural Characterization of Semiconductor Materials and Devices Using Combined Focused Ion Beam and Transmission Electron Microscopy Techniques

1998 ◽  
Vol 523 ◽  
Author(s):  
R. Hull ◽  
D. Dunn
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Semiconductor Materials ◽  
Tomographic Image Reconstruction ◽  
Transmission Electron ◽  
Nanoscale Imaging ◽  
Imaging And Spectroscopy ◽  
Microscopy Techniques

AbstractWe describe novel techniques which extend the range of available nanostructural characterization capability for semiconductor materials and devices. These techniques combine high spatial resolution imaging and spectroscopy using transmission electron microscopy (TEM) and focused ion beam (FIB) microscopy. Specific capabilities described include nanoscale imaging of dopant distributions, ultra-high resolution secondary ion mass spectroscopy (SIMS) and tomographic image reconstruction.

scholarly journals Gold-substituted Silver-intensified Peroxidase Immunolabeling for FIB-SEM Imaging

2019 ◽  
Vol 67 (5) ◽  
pp. 351-360
Author(s):  
Adrian Boey ◽  
Vasily Rybakin ◽  
Dharamdajal Kalicharan ◽  
Katlijn Vints ◽  
Natalia V. Gounko
Keyword(s):  
Electron Microscopy ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Three Dimensional ◽  
Ultrastructural Organization ◽  
High Throughput Analysis ◽  
Detection Techniques ◽  
New Instrumentation ◽  
Technical Issues ◽  
Microscopy Techniques

Modern electron microscopy offers a wide variety of tools to investigate the ultrastructural organization of cells and tissues and to accurately pinpoint intracellular localizations of macromolecules of interest. New volumetric electron microscopy techniques and new instrumentation provide unique opportunities for high-throughput analysis of comparatively large volumes of tissue and their complete reconstitution in three-dimensional (3D) electron microscopy. However, due to a variety of technical issues such as the limited penetration of label into the tissue, low antigen preservation, substantial electron density of secondary detection reagents, and many others, the adaptation of immuno-detection techniques for use with such 3D imaging methods as focused ion beam–scanning electron microscopy (FIB-SEM) has been challenging. Here, we describe a sample preparation method for 3D FIB-SEM, which results in an optimal preservation and staining of ultrastructural details at a resolution necessary for tracing immunolabeled neuronal structures and detailed reconstruction of synapses. This technique is applicable to neuronal and non-neuronal cells, tissues, and a wide variety of antigens.

Growth Directions of Precipitates in the Al–Si–Mg–Hf Alloy Using Combined EBSD and FIB 3D-Reconstruction Techniques

2015 ◽  
Vol 21 (3) ◽  
pp. 588-593 ◽  
Author(s):  
Xueli Wang ◽  
Yuan Xing ◽  
Huilan Huang ◽  
Yanjun Li ◽  
Zhihong Jia ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Three Dimensional ◽  
Normal Direction ◽  
Electron Backscattered Diffraction ◽  
3D Microstructure ◽  
Microscopy Techniques ◽  
Scanning Electron ◽  
Al Matrix

AbstractNanobelt-like precipitates in an Al–Si–Mg–Hf alloy were studied using electron backscattered diffraction (EBSD) and focused ion beam (FIB) scanning electron microscopy techniques. One grain of the Al matrix with a near [111] normal direction was identified by EBSD and the three-dimensional (3D) microstructure of nanobelt-like precipitates in this grain was studied using 3D-FIB. Ten growth directions of the nanobelt-like precipitates in the grain were identified.

scholarly journals Correlative three-dimensional super-resolution and block face electron microscopy of whole vitreously frozen cells

2019 ◽  
Author(s):  
David P. Hoffman ◽  
Gleb Shtengel ◽  
C. Shan Xu ◽  
Kirby R. Campbell ◽  
Melanie Freeman ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Three Dimensional ◽  
Super Resolution ◽  
Field Of View ◽  
Whole Cells ◽  
Workflow Optimization ◽  
Fluorescence And Electron Microscopy ◽  
Block Face

AbstractLiving cells function through the spatial compartmentalization of thousands of distinct proteins serving a multitude of diverse biochemical needs. Correlative super-resolution (SR) fluorescence and electron microscopy (EM) has emerged as a pathway to directly view nanoscale protein relationships to the underlying global ultrastructure, but has traditionally suffered from tradeoffs of structure preservation, fluorescence retention, resolution, and field of view. We developed a platform for three-dimensional correlative cryogenic SR and focused ion beam milled block-face EM across entire vitreously frozen cells that addresses these issues by preserving native ultrastructure and enabling independent SR and EM workflow optimization. Application to a variety of biological systems revealed a number of unexpected protein-ultrastructure relationships and underscored the value of a comprehensive multimodal view of ultrastructural variability across whole cells.

Failure Analysis Using Voltage Contrast and Ebic

1998 ◽  
Vol 523 ◽  
Author(s):  
Larry Rice ◽  
Wei Chen
Keyword(s):  
Electron Microscopy ◽  
Failure Analysis ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Sem Analysis ◽  
Silicon On Insulator ◽  
Traditional Use ◽  
Critical Dimensions ◽  
Voltage Contrast ◽  
Microscopy Techniques

AbstractAs ULSI device critical dimensions continue to shrink to submicron sizes, electron microscopy techniques such as electron beam induced current (EBIC) and voltage contrast are finding more applications towards pinpointing failure sites for subsequent cross sectioning or deprocessing. In addition to the traditional use of EBIC for junction delineation, EBIC has been applied to locate leakage sites in capacitor structures and silicon-on-insulator (SOI) devices as well. Similarly, voltage contrast has been applied to identify single or multiple opens in via chains which consist of thousands of vias. In addition to a brief revisit of the basic principles of EBIC and voltage contrast, focus will be placed on the application of EBIC and voltage contrast in failure analysis of semiconductor devices. Examples of using voltage contrast combined with precision cross section focused ion beam (XFIB) for identifying the failure mechanism of 0.8μm vias will be presented. Also, the use of EBIC for identifying leakage sites in SOI and bipolar devices and subsequent FIB/scanning electron microscopy (SEM) analysis will be presented.

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