Toward Site-Specific Dopant Contrast in Scanning Electron Microscopy

2014 ◽  
Vol 20 (4) ◽  
pp. 1312-1317 ◽  
Author(s):  
Zdena Druckmüllerová ◽  
Miroslav Kolíbal ◽  
Tomáš Vystavěl ◽  
Tomáš Šikola
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Sample Preparation ◽  
Ion Beam ◽  
Three Dimensions ◽  
Dead Layer ◽  
Site Specific ◽  
Transmission Electron ◽  
Buried Layers ◽  
Scanning Electron

AbstractSince semiconductor devices are being scaled down to dimensions of several nanometers there is a growing need for techniques capable of quantitative analysis of dopant concentrations at the nanometer scale in all three dimensions. Imaging dopant contrast by scanning electron microscopy (SEM) is a very promising method, but many unresolved issues hinder its routine application for device analysis, especially in cases of buried layers where site-specific sample preparation is challenging. Here, we report on optimization of site-specific sample preparation by the focused Ga ion beam (FIB) technique that provides improved dopant contrast in SEM. Similar to FIB lamella preparation for transmission electron microscopy, a polishing sequence with decreasing ion energy is necessary to minimize the thickness of the electronically dead layer. We have achieved contrast values comparable to the cleaved sample, being able to detect dopant concentrations down to 1×1016 cm−3. A theoretical model shows that the electronically dead layer corresponds to an amorphized Si layer formed during ion beam polishing. Our results also demonstrate that contamination issues are significantly suppressed for FIB-treated samples compared with cleaved ones.

scholarly journals Electron Microscopy Techniques for Investigating Structure and Composition of Hair-Cell Stereociliary Bundles

2021 ◽  
Vol 9 ◽  
Author(s):  
Maryna V. Ivanchenko ◽  
Artur A. Indzhykulian ◽  
David P. Corey
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Sample Preparation ◽  
Protein Localization ◽  
Imaging Techniques ◽  
Ion Beam ◽  
Immunogold Labeling ◽  
Sensory Cells ◽  
Optimal Sample ◽  
Scanning Electron

Hair cells—the sensory cells of the vertebrate inner ear—bear at their apical surfaces a bundle of actin-filled protrusions called stereocilia, which mediate the cells’ mechanosensitivity. Hereditary deafness is often associated with morphological disorganization of stereocilia bundles, with the absence or mislocalization within stereocilia of specific proteins. Thus, stereocilia bundles are closely examined to understand most animal models of hereditary hearing loss. Because stereocilia have a diameter less than a wavelength of light, light microscopy is not adequate to reveal subtle changes in morphology or protein localization. Instead, electron microscopy (EM) has proven essential for understanding stereocilia bundle development, maintenance, normal function, and dysfunction in disease. Here we review a set of EM imaging techniques commonly used to study stereocilia, including optimal sample preparation and best imaging practices. These include conventional and immunogold transmission electron microscopy (TEM) and scanning electron microscopy (SEM), as well as focused-ion-beam scanning electron microscopy (FIB-SEM), which enables 3-D serial reconstruction of resin-embedded biological structures at a resolution of a few nanometers. Parameters for optimal sample preparation, fixation, immunogold labeling, metal coating and imaging are discussed. Special attention is given to protein localization in stereocilia using immunogold labeling. Finally, we describe the advantages and limitations of these EM techniques and their suitability for different types of studies.

scholarly journals Applications of Scanning Electron Microscopy Using Secondary and Backscattered Electron Signals in Neural Structure

2021 ◽  
Vol 15 ◽  
Author(s):  
Daisuke Koga ◽  
Satoshi Kusumi ◽  
Masahiro Shibata ◽  
Tsuyoshi Watanabe
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Serial Section ◽  
Imaging Technique ◽  
Ion Beam ◽  
Three Dimensions ◽  
Neural Structure ◽  
Array Tomography ◽  
3D Surface ◽  
Scanning Electron

Scanning electron microscopy (SEM) has contributed to elucidating the ultrastructure of bio-specimens in three dimensions. SEM imagery detects several kinds of signals, of which secondary electrons (SEs) and backscattered electrons (BSEs) are the main electrons used in biological and biomedical research. SE and BSE signals provide a three-dimensional (3D) surface topography and information on the composition of specimens, respectively. Among the various sample preparation techniques for SE-mode SEM, the osmium maceration method is the only approach for examining the subcellular structure that does not require any reconstruction processes. The 3D ultrastructure of organelles, such as the Golgi apparatus, mitochondria, and endoplasmic reticulum has been uncovered using high-resolution SEM of osmium-macerated tissues. Recent instrumental advances in scanning electron microscopes have broadened the applications of SEM for examining bio-specimens and enabled imaging of resin-embedded tissue blocks and sections using BSE-mode SEM under low-accelerating voltages; such techniques are fundamental to the 3D-SEM methods that are now known as focused ion-beam SEM, serial block-face SEM, and array tomography (i.e., serial section SEM). This technical breakthrough has allowed us to establish an innovative BSE imaging technique called section-face imaging to acquire ultrathin information from resin-embedded tissue sections. In contrast, serial section SEM is a modern 3D imaging technique for creating 3D surface rendering models of cells and organelles from tomographic BSE images of consecutive ultrathin sections embedded in resin. In this article, we introduce our related SEM techniques that use SE and BSE signals, such as the osmium maceration method, semithin section SEM (section-face imaging of resin-embedded semithin sections), section-face imaging for correlative light and SEM, and serial section SEM, to summarize their applications to neural structure and discuss the future possibilities and directions for these methods.

A Method for Preparation of Site-Specific Multiple Samples of Semiconductor Material for Transmission Electron Microscopy

2001 ◽  
Vol 7 (S2) ◽  
pp. 948-949
Author(s):  
R. S. Rai ◽  
S. Bagchi ◽  
L. Duncan ◽  
L. Prabhu ◽  
J. Beck ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Sample Preparation ◽  
Ion Beam ◽  
Preparation Technique ◽  
Cross Sectional ◽  
Site Specific ◽  
New Approach ◽  
Area Of Interest ◽  
Transmission Electron

In recent years, the availability of focused ion beam (FIB) milling systems has given a much-needed boost for transmission electron microscopy (TEM) as a technique for site-specific analysis. Much progress has been made in the area of site-specific cross-sectional and planar TEM sample preparation techniques. However, a continuing need exists to reduce the sample preparation time, in order to improve TEM cycle time for better support of process development, yield improvement and production in a high-volume industrial environment. Thus, a faster TEM sample preparation technique is always desirable to meet this demand. A new approach to TEM sample preparation is described in this paper.Following the new approach developed in the present work, one can prepare on a single TEM grid at least two different cross-sectional samples of site-specific device structures or up to four different cross-sectional samples of blanket films. Two different samples, each containing an area of interest near the center, are cleaved or cut to a width of about 1.25 mm; these samples may be from two separate locations of a wafer, or from two different wafers where TEM analyses are required.

scholarly journals Quick Sample Preparation and EFTEM Elemental Characterization of FAB Based Defects

2008 ◽  
Vol 16 (3) ◽  
pp. 52-53
Author(s):  
C.T. Schamp ◽  
B.T. Valdez ◽  
J. Gazda
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Electron Microscope ◽  
Sample Preparation ◽  
Atomic Weight ◽  
Elemental Mapping ◽  
Transmission Electron ◽  
Elemental Characterization ◽  
Scanning Electron

A purpose of microscopy is to magnify and enhance contrast between different regions of a sample, whether those regions may be different structures, different orientations of the same structure, regions of different atomic weight, or different chemistries. In the present case, elemental mapping in the energy filtered transmission electron microscope (EFTEM) is used to enhance contrast between elements in an apparent bundle of fibers previously seen through scanning electron microscopy (SEM) and a particle that appears to be a catalytic source for the fibers.

scholarly journals Protocols for Generating Surfaces and Measuring 3D Organelle Morphology Using Amira

Cells ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 65
Author(s):  
Edgar Garza-Lopez ◽  
Zer Vue ◽  
Prasanna Katti ◽  
Kit Neikirk ◽  
Michelle Biete ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
3D Images ◽  
Technological Advances ◽  
Transmission Electron ◽  
Visualization Software ◽  
Organelle Morphology ◽  
Scanning Electron

High-resolution 3D images of organelles are of paramount importance in cellular biology. Although light microscopy and transmission electron microscopy (TEM) have provided the standard for imaging cellular structures, they cannot provide 3D images. However, recent technological advances such as serial block-face scanning electron microscopy (SBF-SEM) and focused ion beam scanning electron microscopy (FIB-SEM) provide the tools to create 3D images for the ultrastructural analysis of organelles. Here, we describe a standardized protocol using the visualization software, Amira, to quantify organelle morphologies in 3D, thereby providing accurate and reproducible measurements of these cellular substructures. We demonstrate applications of SBF-SEM and Amira to quantify mitochondria and endoplasmic reticulum (ER) structures.

scholarly journals Practices for Measuring 3D Organelle Morphology and Generating Surfaces with Amira

2021 ◽  
Author(s):  
Edgar Garza Lopez ◽  
Zer Vue ◽  
Prasanna Katti ◽  
Kit Neikirk ◽  
Michelle Biete ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Technological Advances ◽  
Transmission Electron ◽  
Visualization Software ◽  
Block Face ◽  
Organelle Morphology ◽  
Scanning Electron

Analysis of 3D structures is of paramount importance in cellular biology. Although light microscopy and transmission electron microscopy (TEM) have remained staples for imaging cellular structures, they lack the ability to image in 3D. However, recent technological advances, such as serial block-face scanning electron microscopy (SBF-SEM) and focused ion beam scanning electron microscopy (FIB-SEM), have allowed researchers to observe cellular ultrastructure in 3D. Here, we propose a standardized protocol using the visualization software Amira to quantify organelle morphologies in 3D; this method allows researchers to produce accurate and reproducible measurements of cellular structure characteristics. We demonstrate this applicability by utilizing SBF-SEM and Amira to quantify mitochondria and endoplasmic reticulum (ER) structures.

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