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

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.

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Toward Site-Specific Dopant Contrast in Scanning Electron Microscopy

Microscopy and Microanalysis ◽

10.1017/s1431927614000968 ◽

2014 ◽

Vol 20 (4) ◽

pp. 1312-1317 ◽

Cited By ~ 1

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.

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Influence of sample preparation on the multi scale structure of sand-clay mixtures

E3S Web of Conferences ◽

10.1051/e3sconf/20199201007 ◽

2019 ◽

Vol 92 ◽

pp. 01007

Author(s):

Kexin Yin ◽

Anne-Laure Fauchille ◽

Khaoula Othmani ◽

Giulio Sciarra ◽

Panagiotis Kotronis ◽

...

Keyword(s):

Electron Microscopy ◽

Scanning Electron Microscopy ◽

Sample Preparation ◽

Imaging Techniques ◽

Environmental Scanning Electron Microscopy ◽

Scale Structure ◽

Homogeneous Microstructure ◽

Direct Shear Tests ◽

Multi Scale ◽

Scanning Electron

This paper focuses on the influence of sample preparation on the multi scale structure of sand-clay mixtures. Three different protocols to mix silica and kaolinite were tested in the laboratory to identify the one providing the most homogeneous microstructure. From the macroscopic to the microscopic scales, optical observation, 3D X-ray tomography, 2D scanning electron microscopy (SEM) and 2D environmental scanning electron microscopy (ESEM) were carried out on wet and dry samples. This paper provides a first insight on the mechanisms of sand clay mixing from the cm to μm scale. Preliminary results demonstrate that the microstructures of the samples prepared by the three procedures have similar macroporosities based on imaging techniques. However, the preparation which consists in mixing the sand firstly, followed by water and clay provides a more homogeneous microstructure with silica grains well-surrounded by an oriented clay layering, probably due to a geometrical effect. Understanding the formation of the oriented clay layering brings microstructural features that will help to better explain the grain displacements and rotations during direct shear tests, the behaviour at the pile sand-clay soil interfaces and to formulate sand clay microstructure models.

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Oxygen plasma focused ion beam scanning electron microscopy for biological samples

10.1101/457820 ◽

2018 ◽

Cited By ~ 1

Author(s):

Gorelick Sergey ◽

Korneev Denis ◽

Handley Ava ◽

Gervinskas Gediminas ◽

Oorschot Viola ◽

...

Keyword(s):

Electron Microscopy ◽

Scanning Electron Microscopy ◽

Sample Preparation ◽

Biological Samples ◽

Oxygen Plasma ◽

Focused Ion Beam ◽

Ion Beam ◽

Light And Electron Microscopy ◽

Beam Scanning ◽

Scanning Electron

AbstractOver the past decade, gallium Focused Ion Beam-Scanning Electron Microscopy (FIB-SEM) has been established as a key technology for cellular tomography. The utility of this approach, however, is severely limited both by throughput and the limited selection of compatible sample preparation protocols. Here, we address these limitations and present oxygen plasma FIB (O-PFIB) as a new and versatile tool for cellular FIB-SEM tomography. Oxygen displays superior resin compatibility to other ion beams and produces curtain-free surfaces with minimal polishing. Our novel approach permits more flexible sample preparation and 30% faster data collection when compared to using gallium ion sources. We demonstrate this alternative FIB is applicable to a variety of embedding procedures and biological samples including brain tissue and whole organisms. Finally, we demonstrate the use of O-PFIB to produce targeted FIB-SEM tomograms through fiducial free en-block correlative light and electron microscopy.

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Ultrastructural Features of Normal and Diseased Hair Revealed by Ion Beam Etching and Freeze Fracture

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100115419 ◽

1975 ◽

Vol 33 ◽

pp. 358-359

Author(s):

M. Spector ◽

A. C. Brown

Keyword(s):

Electron Microscopy ◽

Scanning Electron Microscopy ◽

Ectodermal Dysplasia ◽

Ion Beam ◽

Freeze Fracture ◽

Ion Current ◽

Ion Beam Etching ◽

Pili Torti ◽

Argon Ion ◽

Scanning Electron

Ion beam etching and freeze fracture techniques were utilized in conjunction with scanning electron microscopy to study the ultrastructure of normal and diseased human hair. Topographical differences in the cuticular scale of normal and diseased hair were demonstrated in previous scanning electron microscope studies. In the present study, ion beam etching and freeze fracture techniques were utilized to reveal subsurface ultrastructural features of the cuticle and cortex.Samples of normal and diseased hair including monilethrix, pili torti, pili annulati, and hidrotic ectodermal dysplasia were cut from areas near the base of the hair. In preparation for ion beam etching, untreated hairs were mounted on conducting tape on a conducting silicon substrate. The hairs were ion beam etched by an 18 ky argon ion beam (5μA ion current) from an ETEC ion beam etching device. The ion beam was oriented perpendicular to the substrate. The specimen remained stationary in the beam for exposures of 6 to 8 minutes.

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Sample Preparation for Translucent and Scanning Electron Microscopy: New Leica Microsystems Coaters

Science and innovation ◽

10.15407/scine10.02.050 ◽

2014 ◽

Vol 10 (2) ◽

pp. 50-54

Author(s):

S.V. Niemova ◽

Keyword(s):

Electron Microscopy ◽

Scanning Electron Microscopy ◽

Sample Preparation ◽

Scanning Electron

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Microscopy Methods for Biofilm Imaging: Focus on SEM and VP-SEM Pros and Cons

Biology ◽

10.3390/biology10010051 ◽

2021 ◽

Vol 10 (1) ◽

pp. 51

Author(s):

Michela Relucenti ◽

Giuseppe Familiari ◽

Orlando Donfrancesco ◽

Maurizio Taurino ◽

Xiaobo Li ◽

...

Keyword(s):

Electron Microscopy ◽

Scanning Electron Microscopy ◽

Ion Beam ◽

Ruthenium Red ◽

Variable Pressure ◽

Sem Images ◽

Advantages And Disadvantages ◽

Pros And Cons ◽

Microscopy Techniques ◽

Scanning Electron

Several imaging methodologies have been used in biofilm studies, contributing to deepening the knowledge on their structure. This review illustrates the most widely used microscopy techniques in biofilm investigations, focusing on traditional and innovative scanning electron microscopy techniques such as scanning electron microscopy (SEM), variable pressure SEM (VP-SEM), environmental SEM (ESEM), and the more recent ambiental SEM (ASEM), ending with the cutting edge Cryo-SEM and focused ion beam SEM (FIB SEM), highlighting the pros and cons of several methods with particular emphasis on conventional SEM and VP-SEM. As each technique has its own advantages and disadvantages, the choice of the most appropriate method must be done carefully, based on the specific aim of the study. The evaluation of the drug effects on biofilm requires imaging methods that show the most detailed ultrastructural features of the biofilm. In this kind of research, the use of scanning electron microscopy with customized protocols such as osmium tetroxide (OsO4), ruthenium red (RR), tannic acid (TA) staining, and ionic liquid (IL) treatment is unrivalled for its image quality, magnification, resolution, minimal sample loss, and actual sample structure preservation. The combined use of innovative SEM protocols and 3-D image analysis software will allow for quantitative data from SEM images to be extracted; in this way, data from images of samples that have undergone different antibiofilm treatments can be compared.

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