Electron Microscopy Sample Preparation Research Articles

Thickness-dependent stabilization of tetragonal ZrO2 in oxidized zirconium

Here we describe a study on the effect of transmission electron microscopy sample preparation on the stability of the tetragonal ZrO2 phase, as determined by precession electron diffraction. It was found that sample thickness, and thus corresponding stress remaining within the sample, can significantly affect the percentage of tetragonal phase ZrO2 recorded in the oxide layers. The microscopy-based finding is complemented by density functional theory calculations, confirming that there exists a crossover in energy between the two phases that depends on compressive strain. These findings further confirm that the significant portion of tetragonal phase is the result of stress stabilization.

Intracellular pathway and subsequent transformation of hydroxyapatite nanoparticles in the SAOS-2 osteoblast cell line.

Internalization of hydroxyapatite nanoparticles in SAOS-2 osteoblasts for 2 and 24 h was investigated in vitro using 5 and 50 µg/mL nanoparticles in culture medium. No cytotoxic effects were observed in a PrestoBlue viability assay. Focused ion beam-scanning electron microscopy and transmission electron microscopy were used to study nanoparticle trafficking inside cells and to characterize the physicochemical properties of the remodeled nanoparticles. Nanoparticles were actively internalized by cells and maintained in intracellular membrane-bound compartments. Dissolution of hydroxyapatite nanoparticles was observed inside phagolysosome in all samples. After 24 h of internalization in cell culture assays, reprecipitation of calcium phosphate minerals was observed in membrane-bound compartments in 5 and 50 µg/mL samples. Compared to the original nanoparticles, the reprecipitated calcium phosphate phase presented a different morphology, structure, and chemical composition. Two sample preparation methods were used and confirmed that reprecipitation of the calcium phosphate crystallites occurred in the intracellular environment and not during electron microscopy sample preparation. Reprecipitation of calcium phosphate prevented the release of large amounts of calcium and phosphate ions inside the cells. This phenomenon may be linked to physiological processes in the cell that control the concentration and trafficking of intracellular calcium ions, which are highly controlled by cells. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 428-439, 2018.

Novel Method for Preparing Transmission Electron Microscopy Samples of Micrometer-Sized Powder Particles by Using Focused Ion Beam.

The preparation of transmission electron microscopy (TEM) samples from powders is quite difficult and challenging. For powders with particles in the 1-5 μm size range, it is especially difficult to select an adequate sample preparation technique. Epoxy is commonly used to bind powder, but drawbacks, such as differential milling originating from unequal milling rates between the epoxy and powder, remain. We propose a new, simple method for preparing TEM samples. This method is especially useful for powders with particles in the 1-5 μm size range that are vulnerable to oxidation. The method uses solder as an embedding agent together with focused ion beam (FIB) milling. The powder was embedded in low-temperature solder using a conventional hot-mounting instrument. Subsequently, FIB was used to fabricate thin TEM samples via the lift-out technique. The solder proved to be more effective than epoxy in producing thin TEM samples with large areas. The problem of differential milling was mitigated, and the solder binder was more stable than epoxy under an electron beam. This methodology can be applied for preparing TEM samples from various powders that are either vulnerable to oxidation or composed of high atomic number elements.

Electron microscopy using the genetically encoded APEX2 tag in cultured mammalian cells.

Electron microscopy (EM) is the premiere technique for high-resolution imaging of cellular ultrastructure. Unambiguous identification of specific proteins or cellular compartments in electron micrographs, however, remains challenging because of difficulties in delivering electron-dense contrast agents to specific subcellular targets within intact cells. We recently reported enhanced ascorbate peroxidase 2 (APEX2) as a broadly applicable genetic tag that generates EM contrast on a specific protein or subcellular compartment of interest. This protocol provides guidelines for designing and validating APEX2 fusion constructs, along with detailed instructions for cell culture, transfection, fixation, heavy-metal staining, embedding in resin, and EM imaging. Although this protocol focuses on EM in cultured mammalian cells, APEX2 is applicable to many cell types and contexts, including intact tissues and organisms, and is useful for numerous applications beyond EM, including live-cell proteomic mapping. This protocol, which describes procedures for sample preparation from cell monolayers and cell pellets, can be completed in 10 d, including time for APEX2 fusion construct validation, cell growth, and solidification of embedding resins. Notably, the only additional steps required relative to a standard EM sample preparation are cell transfection and a 2- to 45-min staining period with 3,3-diaminobenzidine (DAB) and hydrogen peroxide (H2O2).

Quantitating morphological changes in biological samples during scanning electron microscopy sample preparation with correlative super-resolution microscopy.

Sample preparation is critical to biological electron microscopy (EM), and there have been continuous efforts on optimizing the procedures to best preserve structures of interest in the sample. However, a quantitative characterization of the morphological changes associated with each step in EM sample preparation is currently lacking. Using correlative EM and superresolution microscopy (SRM), we have examined the effects of different drying methods as well as osmium tetroxide (OsO4) post-fixation on cell morphology during scanning electron microscopy (SEM) sample preparation. Here, SRM images of the sample acquired under hydrated conditions were used as a baseline for evaluating morphological changes as the sample went through SEM sample processing. We found that both chemical drying and critical point drying lead to a mild cellular boundary retraction of ~60 nm. Post-fixation by OsO4 causes at least 40 nm additional boundary retraction. We also found that coating coverslips with adhesion molecules such as fibronectin prior to cell plating helps reduce cell distortion from OsO4 post-fixation. These quantitative measurements offer useful information for identifying causes of cell distortions in SEM sample preparation and improving current procedures.

Lattice reorientation in tetragonal PMN-PT thin film induced by focused ion beam preparation for transmission electron microscopy

Focused ion beam sample preparation for transmission electron microscopy (TEM) can induce relaxation mechanisms in epitaxial thin films. Here, we describe a relaxation mechanism that can occur in materials having a tetragonal structure. We investigated the lattice structure of a 600 nm thick 0.4[Pb(Mg1/3Nb2/3)O3]−0.6[PbTiO3] layer grown by epitaxy on (110) GdScO3 substrate using geometrical phase analysis applied to high resolution TEM images. The lattice mismatch at the interface is expected to favor the formation of c-domains. However, it was measured that the out-of-plane lattice parameter can decrease abruptly along the growth direction and the transition depends on the thickness of the TEM lamella. Different observations indicate that the crystal flipped by 90° following the preparation of the sample, so that the c-axis is oriented in the thinning direction. Such a mechanism can easily lead to misinterpretations and might happen in other materials with a similar structure.

Intracerebral Injections and Ultrastructural Analysis of High-Pressure Frozen Brain Tissue.

Intracerebral injections are an invasive method to bypass the blood brain barrier and are widely used to study molecular and cellular mechanisms of the central nervous system. The administered substances are injected directly at the site of interest, executing their effect locally. By combining injections in the rat brain with state-of-the-art electron microscopy, subtle changes in ultrastructure of the nervous tissue can be detected prior to overt damage or disease. The protocol presented here involves stereotactic injection into the corpus callosum of Lewis rats and the cryopreparation of freshly dissected tissue for electron microscopy. The localization of the injection site in tissue sections during the sample preparation for transmission electron microscopy is explained and possible artifacts of the method are indicated. With the help of this powerful combination of injections and electron microscopy, subtle effects of the applied substances on the biology of neural cells can be identified and monitored over time. © 2017 by John Wiley & Sons, Inc.

Scanning Electron Microscopy Sample Preparation and Imaging.

Scanning electron microscopes allow us to reach magnifications of 20-130,000× and resolve compositional and topographical images with intense detail. These images are created by bombarding a sample with electrons in a focused manner to generate a black and white image from the electrons that bounce off of the sample. The electrons are detected using positively charged detectors. Scanning electron microscopy permits three-dimensional imaging of desiccated specimens or wet cells and tissues by using variable pressure chambers. SEM ultrastructural analysis and intracellular imaging supplement light microscopy for molecular profiling of prokaryotes, plants, and mammals. This chapter demonstrates how to prepare and image samples that are (a) desiccated and conductive, (b) desiccated and nonconductive but coated with an electron conductive film using a gold sputter coater, and

Electron Microscopy of Ebola Virus-Infected Cells.

Ebola virus (EBOV) replicates in host cells, where both viral and cellular components show morphological changes during the process of viral replication from entry to budding. These steps in the replication cycle can be studied using electron microscopy (EM), including transmission electron microscopy (TEM) and scanning electron microscopy (SEM), which is one of the most useful methods for visualizing EBOV particles and EBOV-infected cells at the ultrastructural level. This chapter describes conventional methods for EM sample preparation of cultured cells infected with EBOV.

Preserving the photoswitching ability of standard fluorescent proteins for correlative in-resin super-resolution and electron microscopy.

There are many different correlative light and electron microscopy (CLEM) techniques available. The use of super-resolution microscopy in CLEM is an emerging application and while offering the obvious advantages of improved resolution in the fluorescence image, and therefore more precise correlation to electron microscopy (EM) ultrastructure, it also presents new challenges. Choice of fluorophore, method of fixation, and timing of the fluorescence imaging are critical to the success of super-resolution CLEM and the relative importance, and technical difficulty, of each of these factors depends on the type of super-resolution microscopy being employed. This chapter details the method we developed for in-resin super-resolution CLEM using single molecule localization microscopy (SMLM) with standard fluorescent proteins (e.g., GFP and mVenus). The key to this approach is being able to preserve not only the fluorescence, but also, and more importantly, the photoswitching ability of the fluorescent proteins throughout the EM sample preparation procedure. Cells are cryofixed using high pressure freezingfor optimal structural preservation and then freeze substituted in tannic acid, which preserves the photoswitching ability of the fluorescent proteins and is essential for high-quality SMLM imaging. Resin sections are then imaged using SMLM, achieving a structural resolution of 40-50nm and a localization precision of ∼17nm, followed by transmission electron microscopy. This produces high quality correlative images without the use of specialized fluorescent proteins or antibodies.

Electron microscopic investigation and elemental analysis of titanium dioxide in sun lotion.

The objective of this research was to determine the size, shape and aggregation of titanium dioxide (TiO2 ) particles which are used in sun lotion as UV-blocker. Overall, six sunscreens from various suppliers and two reference substances were analysed by electron microscopy (EM) techniques in combination with energy dispersive X-ray spectroscopy (EDS). Because of a high fat content in sun lotion, it was impossible to visualize the TiO2 particles without previous EM sample preparation. Different defatting methods for TiO2 from sun screens were tested. A novel sample preparation method was developed which allowed the characterization of TiO2 particles with the help of EM and EDS. Aggregates of titanium dioxide with the size of primary particles varying between 15 and 40 nm were observed only in five products. In the sun lotion with the highest SPF, only few small aggregates were found. In the sun screen with the lowest SPF, the largest aggregates of TiO2 particles were detected with sizes up to 1.6 μm. In one of the sun lotions, neither TiO2 nor ZnO was found in spite of the labelling. Instead, approx. 500 nm large diamond-shaped particles were observed. These particles are composed of an organic material as only carbon was detected by EDS. A novel defatting method for sample preparation of titanium dioxide nanoparticles used in sun cosmetics was developed. This method was applied to six different sun lotions with SPF between 30 and 50+. TiO2 particles were found in only five sunscreens. The sizes of the primary particles were below 100 nm and, according to the EU Cosmetic Regulation, have to be listed on the package with the term 'nano'.

Cross-Sectional Transmission Electron Microscopy Sample Preparation of Soldering Joint Using Ultramicrotomy

Many studies have reported the interfacial reaction of Sn-Ag/ electroless nickel immersion gold (ENIG). Various analysis techniques, such as scanning electron microscopy, electron probe microanalyzer, and transmission electron microscopy (TEM), have been used to examine interfacial reaction. Among them, TEM equipped with analytical attachments offers the most complete tool for characterizing the formation of intermetallic compounds (IMCs) at the interface. However, solders are typically soft and ductile, while IMCs tend to be quite hard and brittle, which has limited the level of success in preparing TEM samples. We utilized ultramicrotomy since it might offer a reasonable and relatively quick alternative in this case. Ultramicrotomy has been used for the preparation of TEM specimens from biological materials and extending a range of materials including polymers, coating layer, particles, and even metals (Becker & Bange, 1993; Glanvill, 1995; Howell et al., 1995). TEM sample making to section hard materials by ultramicrotomy are not new. Besides, it has been traditionally considered that the mechanical damage during sectioning by diamond knife might hinder unambiguous specimen characterization. However, this technique also has many advantages such as no ion beam irradiation damage, no chemical mixing, no differential thinning rate and the ease of preparation of many serial sections with large, thin areas of uniform thickness in a relatively short time (Quintana, 1997). The increasing acceptability of ultramicrotomy in materials science is evidenced by a number of recent paper giving results for different hard materials (Hwang & Suganuma, 2003).

Fabrication of high aspect ratio AFM probes with different materials inspired by TEM “lift-out” method

The most commonly used materials in all commercially available high-aspect-ratio (HAR) nanowire's (NW) tips are made of silicon and carbon nanotube which limit their applications in other types of atomic force microscopy (AFM), such as conducting AFM and magnetic force microscope. Therefore, a simple process inspired by cross-sectional transmission electron microscopy sample preparation method was used to demonstrate the feasibility of fabricating HAR AFM probes, which can easily define the tilt angle of the NW tip with respect to the direction that is normal to the axis of the cantilever to which it is attached by simply tilting the sample stage where the cantilever is placed. This is very important as it enables precise control of the inclination angle of the NW tip and allows the tip to be made perpendicular to the probed surface for scanning with different AFM mounts. Two different tips were fabricated, one attached parallel and the other attached at an angle of 13° with respect to the normal of the cantilever axis. These tips were used to profile the topography of a silicon nanopillar array. Only the probe attached at an angle of 13° allowed mapping of the topography between nanopillars. This is the first successful demonstration of an HAR AFM tip being used to map the topography of a nanopillar array. In addition, the authors also demonstrated that this method can be extended to fabricate HAR AFM tips of different materials such as copper with a slightly modified approach.

Optimized cryo-focused ion beam sample preparation aimed at in situ structural studies of membrane proteins.

While cryo-electron tomography (cryo-ET) can reveal biological structures in their native state within the cellular environment, it requires the production of high-quality frozen-hydrated sections that are thinner than 300nm. Sample requirements are even more stringent for the visualization of membrane-bound protein complexes within dense cellular regions. Focused ion beam (FIB) sample preparation for transmission electron microscopy (TEM) is a well-established technique in material science, but there are only few examples of biological samples exhibiting sufficient quality for high-resolution in situ investigation by cryo-ET. In this work, we present a comprehensive description of a cryo-sample preparation workflow incorporating additional conductive-coating procedures. These coating steps eliminate the adverse effects of sample charging on imaging with the Volta phase plate, allowing data acquisition with improved contrast. We discuss optimized FIB milling strategies adapted from material science and each critical step required to produce homogeneously thin, non-charging FIB lamellas that make large areas of unperturbed HeLa and Chlamydomonas cells accessible for cryo-ET at molecular resolution.

Evaluation of Neon Focused Ion Beam Milling for TEM Sample Preparation

Gallium-based focused ion beams generated from liquid-metal sources are widely used in micromachining and sample preparation for transmission electron microscopy, with well-known drawbacks such as sample damage and contamination. In this work, an alternative (neon) focused ion beam generated by a gas field-ionization source is evaluated for the preparation of electron-transparent specimens. To do so, electron-transparent sections of Si and an Al alloy are prepared with both Ga and Ne ion beams for direct comparison. Diffraction-contrast imaging and energy dispersive x-ray spectroscopy are used to evaluate the relative damage induced by the two beams, and cross-sections of milled trenches are examined to compare the implantation depth with theoretical predictions from Monte Carlo simulations. Our results show that for the beam voltages and materials systems investigated, Ne ion beam milling does not significantly reduce the focused ion beam induced artefacts. However, the Ne ion beam does enable more precise milling and may be of interest in cases where Ga contamination cannot be tolerated.

Large-scale Scanning Transmission Electron Microscopy (Nanotomy) of Healthy and Injured Zebrafish Brain

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution electron microscopy. Others and we previously applied large scale EM to human skin pancreatic islets, tissue culture and whole zebrafish larvae1-7. Here we describe a universally applicable method for tissue-scale scanning EM for unbiased detection of sub-cellular and molecular features. Nanotomy was applied to investigate the healthy and a neurodegenerative zebrafish brain. Our method is based on standardized EM sample preparation protocols: Fixation with glutaraldehyde and osmium, followed by epoxy-resin embedding, ultrathin sectioning and mounting of ultrathin-sections on one-hole grids, followed by post staining with uranyl and lead. Large-scale 2D EM mosaic images are acquired using a scanning EM connected to an external large area scan generator using scanning transmission EM (STEM). Large scale EM images are typically ~ 5 - 50 G pixels in size, and best viewed using zoomable HTML files, which can be opened in any web browser, similar to online geographical HTML maps. This method can be applied to (human) tissue, cross sections of whole animals as well as tissue culture1-5. Here, zebrafish brains were analyzed in a non-invasive neuronal ablation model. We visualize within a single dataset tissue, cellular and subcellular changes which can be quantified in various cell types including neurons and microglia, the brain's macrophages. In addition, nanotomy facilitates the correlation of EM with light microscopy (CLEM)8 on the same tissue, as large surface areas previously imaged using fluorescent microscopy, can subsequently be subjected to large area EM, resulting in the nano-anatomy (nanotomy) of tissues. In all, nanotomy allows unbiased detection of features at EM level in a tissue-wide quantifiable manner.

Large-scale Scanning Transmission Electron Microscopy (Nanotomy) of Healthy and Injured Zebrafish Brain.

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution electron microscopy. Others and we previously applied large scale EM to human skin pancreatic islets, tissue culture and whole zebrafish larvae1-7. Here we describe a universally applicable method for tissue-scale scanning EM for unbiased detection of sub-cellular and molecular features. Nanotomy was applied to investigate the healthy and a neurodegenerative zebrafish brain. Our method is based on standardized EM sample preparation protocols: Fixation with glutaraldehyde and osmium, followed by epoxy-resin embedding, ultrathin sectioning and mounting of ultrathin-sections on one-hole grids, followed by post staining with uranyl and lead. Large-scale 2D EM mosaic images are acquired using a scanning EM connected to an external large area scan generator using scanning transmission EM (STEM). Large scale EM images are typically ~ 5 - 50 G pixels in size, and best viewed using zoomable HTML files, which can be opened in any web browser, similar to online geographical HTML maps. This method can be applied to (human) tissue, cross sections of whole animals as well as tissue culture1-5. Here, zebrafish brains were analyzed in a non-invasive neuronal ablation model. We visualize within a single dataset tissue, cellular and subcellular changes which can be quantified in various cell types including neurons and microglia, the brain's macrophages. In addition, nanotomy facilitates the correlation of EM with light microscopy (CLEM)8 on the same tissue, as large surface areas previously imaged using fluorescent microscopy, can subsequently be subjected to large area EM, resulting in the nano-anatomy (nanotomy) of tissues. In all, nanotomy allows unbiased detection of features at EM level in a tissue-wide quantifiable manner.

Evaluation of neon focused ion beam milling for TEM sample preparation.

Gallium-based focused ion beams generated from liquid-metal sources are widely used in micromachining and sample preparation for transmission electron microscopy, with well-known drawbacks such as sample damage and contamination. In this work, an alternative (neon) focused ion beam generated by a gas field-ionization source is evaluated for the preparation of electron-transparent specimens. To do so, electron-transparent sections of Si and an Al alloy are prepared with both Ga and Ne ion beams for direct comparison. Diffraction-contrast imaging and energy dispersive x-ray spectroscopy are used to evaluate the relative damage induced by the two beams, and cross-sections of milled trenches are examined to compare the implantation depth with theoretical predictions from Monte Carlo simulations. Our results show that for the beam voltages and materials systems investigated, Ne ion beam milling does not significantly reduce the focused ion beam induced artefacts. However, the Ne ion beam does enable more precise milling and may be of interest in cases where Ga contamination cannot be tolerated.

Architectural plasticity of AMPK revealed by electron microscopy and X-ray crystallography.

Mammalian AMP-activated protein kinase (AMPK) acts as an important sensor of cellular energy homeostasis related with AMP/ADP to ATP ratio. The overall architecture of AMPK has been determined in either homotrimer or monomer form by electron microscopy (EM) and X-ray crystallography successively. Accordingly proposed models have consistently revealed a key role of the α subunit linker in sensing adenosine nucleoside binding on the γ subunit and mediating allosteric regulation of kinase domain (KD) activity, whereas there are vital differences in orienting N-terminus of α subunit and locating carbohydrate-binding module (CBM) of β subunit. Given that Mg2+, an indispensable cofactor of AMPK was present in the EM sample preparation buffer however absent when forming crystals, here we carried out further reconstructions without Mg2+ to expectably inspect if this ion may contribute to this difference. However, no essential alteration has been found in this study compared to our early work. Further analyses indicate that the intra-molecular movement of the KD and CBM are most likely due to the flexible linkage of the disordered linkers with the rest portion as well as a contribution from the plasticity in the inter-molecular assembly mode, which might ulteriorly reveal an architectural complication of AMPK.

APT mass spectrometry and SEM data for CdTe solar cells.

Atom probe tomography (APT) data acquired from a CAMECA LEAP 4000 XHR for the CdS/CdTe interface for a non-CdCl2 treated CdTe solar cell as well as the mass spectrum of an APT data set including a GB in a CdCl2-treated CdTe solar cell are presented. Scanning electron microscopy (SEM) data showing the evolution of sample preparation for APT and scanning transmission electron microscopy (STEM) electron beam induced current (EBIC) are also presented. These data show mass spectrometry peak decomposition of Cu and Te within an APT dataset, the CdS/CdTe interface of an untreated CdTe solar cell, preparation of APT needles from the CdS/CdTe interface in superstrate grown CdTe solar cells, and the preparation of a cross-sectional STEM EBIC sample.