Specimen Preparation For Electron Microscopy Research Articles

Automated Specimen Preparation for Electron Microscopy

Automated Specimen Preparation for Electron Microscopy

Lift-out cryo-FIBSEM and cryo-ET reveal the ultrastructural landscape of extracellular matrix.

The extracellular matrix (ECM) serves as a scaffold for cells and plays an essential role in regulating numerous cellular processes, including cell migration and proliferation. Due to limitations in specimen preparation for conventional room-temperature electron microscopy, we lack structural knowledge on how ECM components are secreted, remodeled, and interact with surrounding cells. We have developed a 3D-ECM platform compatible with sample thinning by cryo-focused ion beam milling, the lift-out extraction procedure, and cryo-electron tomography. Our workflow implements cell-derived matrices (CDMs) grown on EM grids, resulting in a versatile tool closely mimicking ECM environments. This allows us to visualize ECM for the first time in its hydrated, native context. Our data reveal an intricate network of extracellular fibers, their positioning relative to matrix-secreting cells, and previously unresolved structural entities. Our workflow and results add to the structural atlas of the ECM, providing novel insights into its secretion and assembly.

Creating Efficient Workflows for Electron Microscopy Laboratories with Automated Specimen Preparation

Abstract Electron microscopy (EM) is essential to the biological and biomedical sciences and clinical diagnostic pathology. Electron microscopy specimen preparation is laborious and time-consuming, with transmission EM (TEM) chemical preparation typically requiring 1–3 days, while volume electron microscopy (vEM) takes 3–5 days of tedious manual reagent exchanges every few minutes or hours. This places a considerable burden on laboratory scientists with the ongoing demand for TEM, and the rapidly growing demand for vEM due to its potential to revolutionize structural biology, connectomics, and related fields. This burden is exacerbated by a shortage of trained electron microscopy scientists as current staff retire, and few enter the workforce. This report provides four case studies to illustrate how automated and faster specimen preparation workflows using mPrep™ Automated Specimen Processors (ASP-1000™ and ASP-2000™, Microscopy Innovations, LLC) free electron microscopy staff in academic and pre-clinical research labs and a clinical pathology laboratory.

Applications of scanning electron microscopy and focused ion beam milling in dental research.

The abilities of scanning electron microscopy (SEM) and focused ion beam (FIB) milling for obtaining high-resolution images from top surfaces, cross-sectional surfaces, and even in three dimensions, are becoming increasingly important for imaging and analyzing tooth structures such as enamel and dentin. FIB was originally developed for material research in the semiconductor industry. However, use of SEM/FIB has been growing recently in dental research due to the versatility of dual platform instruments that can be used as a milling device to obtain low-artifact cross-sections of samples combined with high-resolution images. The advent of the SEM/FIB system and accessories may offer access to previously inaccessible length scales for characterizing tooth structures for dental research, opening exciting opportunities to address many central questions in dental research. New discoveries and fundamental breakthroughs in understanding are likely to follow. This review covers the applications, key findings, and future direction of SEM/FIB in dental research in morphology imaging, specimen preparation for transmission electron microscopy (TEM) analysis, and three-dimensional volume imaging using SEM/FIB tomography.

Optimal Specimen Preparation for Correlative Atom Probe Tomography and Electron Microscopy of Environmentally Sensitive Materials

Optimal Specimen Preparation for Correlative Atom Probe Tomography and Electron Microscopy of Environmentally Sensitive Materials

Twin-jet electropolishing for damage-free transmission electron microscopy specimen preparation of metallic microwires.

A method to prepare TEM specimens from metallic microwires and based on conventional twin-jet electropolishing is introduced. The wire is embedded in an opaque epoxy resin medium and the hardened resin is mechanically polished to reveal the wire on both sides. The resin containing wire is then cut into discs of the appropriate size. The obtained embedded wire is electropolished in a conventional twin-jet electropolishing machine until electron transparency in large areas without radiation damage is achieved.

Ultra-Microtome for the Preparation of TEM Specimens from Battery Cathodes.

With the wide application of ultra-microtome sectioning in the preparation of transmission electron microscopy (TEM) specimens with bio- and organic materials, here, we report an ultra-microtome-based method for the preparation of TEM specimens from cathodes of Li-ion batteries. The ultra-microtome sectioning reduces the sample thickness to tens of nanometers and yields atomic resolution from the core region of particles of hundreds of nanometers. Analysis indicates that the mechanical cross-sectioning introduces no observable microstructural artifacts or structural damage, such as microcracking and nanoporosity. These results demonstrate the high efficiency of the ultra-microtome approach in preparing well-thinned specimens of particulate materials that allow for atomic-scale TEM imaging of a large number of sectioned particles in one single TEM specimen, thereby providing statistically significant results of the TEM analysis.

The need to freeze-Dehydration during specimen preparation for electron microscopy collapses the endothelial glycocalyx regardless of fixation method.

The endothelial glycocalyx covers the luminal surface of the endothelium and plays key roles in vascular function. Despite its biological importance, ideal visualization techniques are lacking. The current study aimed to improve the preservation and subsequent imaging quality of the endothelial glycocalyx. In mice, the endothelial glycocalyx was contrasted with a mixture of lanthanum and dysprosium (LaDy). Standard chemical fixation was compared with high-pressure frozen specimens processed with freeze substitution. Also, isolated brain microvessels and cultured endothelial cells were high-pressure frozen and by transmission soft x-rays, imaged under cryogenic conditions. The endothelial glycocalyx was in some tissues significantly more voluminous from chemically fixed specimens compared with high-pressure frozen specimens. LaDy labeling introduced excessive absorption contrast, which impeded glycocalyx measurements in isolated brain microvessels when using transmission soft x-rays. In non-contrasted vessels, the glycocalyx was not resolved. LaDy-contrasted, cultured brain endothelial cells allowed to assess glycocalyx volume in vitro. Both chemical and cryogenic fixation followed by dehydration lead to substantial collapse of the glycocalyx. Cryogenic fixation without freeze substitution could be a way forward although transmission soft x-ray tomography based solely on amplitude contrast seems unsuitable.

Application of focused ion-beam sampling for sidewall-roughness measurement of free-standing sub-μm objects by atomic force microscopy.

In the present study, a free-standing object-sampling technique for microelectromechanical systems (MEMS) is developed to measure their sidewall surface roughnesses by atomic force microscopy (AFM). For this purpose, a conventional focused ion beam (FIB) sampling technique widely used for cross-sectional transmission electron microscope specimen preparation was applied. The sub-nm-order roughness parameters were quantitatively measured for sidewalls of Si-bridge test samples. The roughness parameters were compared before and after H2 annealing treatment, which induced smoothing of the surface by migration of the Si atoms. The reduction in the surface roughness by a factor of approximately one-third with 60-s H2 annealing was quantitatively evaluated by AFM. The present study confirms that the developed FIB-AFM technique is one potential approach for quantitatively evaluating the surface-roughness parameters on the oblique faces of free-standing objects in MEMS devices.

Direct measurement of TEM lamella thickness in FIB-SEM.

Transmission electron microscope (TEM) specimen preparation by focused ion beam (FIB) milling requires delicate polishing of a thin window of material during the final stages of the procedure. Over or underpolishing is common and requires extra microscope resources to correct. Despite some methods for lamella thickness measurement being available, the majority of users judge the final polishing step subjectively from scanning electron microscope (SEM) images acquired between milling steps. Here we demonstrate successful thickness determination of thin silicon lamellae using calibrated secondary electron detectors in a FIB-SEM dual-beam chamber. Unlike previous thickness measurement methods it does not require long acquisition times, the use of in-chamber scanning transmission electron microscope (STEM) or energy dispersive x-ray spectroscopy detectors. The calibration aligns a SEM image to an electron energy loss spectroscopy (EELS) map of lamella thickness acquired in a TEM. This calibration reveals the greyscale-thickness dependence of two secondary electron SEM detectors: the through-lens detector (TLD) and the in-chamber electron detector (ICE). It was found that lamella thickness estimation for TLD images is accurate for areas thinner than 0.4 t/λ, whilst ICE images are most accurate for areas thicker than 0.5 t/λ up to 1.1 t/λ. The procedure presented here allows objective lamella thickness determination during the final stages of FIB specimen preparation using conventional imaging modes for common secondary electron detectors. LAY DESCRIPTION: Successful analysis of a material in a transmission electron microscope requires very thin windows of the material to be fabricated. Despite the quality of this analysis relying heavily on the thickness of the window, measuring thickness during window fabrication is not common practice. The authors show that it is possible to measure the thickness of the window directly in a focused-ion-beam chamber with a scanning electron microscope without altering the fabrication procedure, and using electron detectors common to most microscopes.

Combined Focused Ion Beam-Ultramicrotomy Method for TEM Specimen Preparation of Porous Fine-Grained Materials.

A new transmission electron microscopy (TEM) specimen preparation method that utilizes a combination of focused ion beam (FIB) methods and ultramicrotomy is demonstrated. This combined method retains the benefit of site-specific sampling by FIB but eliminates ion beam-induced damage except at specimen edges and allows recovery of many consecutive sections. It is best applied to porous and/or fine-grained materials that are amenable to ultramicrotomy but are located in bulk samples that are not. The method is ideal for unique samples from which every specimen is precious, and we demonstrate its utility on fine-grained material from the one-of-a-kind Paris meteorite. Compared with a specimen prepared by conventional FIB methods, the final sections are uniformly thin and free from re-deposition and curtaining artifacts common in FIB specimens prepared from porous, heterogeneous samples.

A novel specimen preparation of porous cathode materials in lithium-ion batteries for high-resolution transmission electron microscopy

Micropores and fissures in cathode materials are associated with deterioration of electrochemical performance of lithium-ion batteries. Phase transformation is responsible for anisotropic volume change and pulverization in conjunction with an expansion of the pores and fissures, occurred dominantly at the outermost cathode particles where lithium ions are repeatedly diffused from parent structure to anode and vice versa. The gallium ion sources, in addition, penetrate into the vacuum and etch the particle in the vicinity of pores and fissures during the cross-sectioning process using focused ion beam. Herein, we propose a noble specimen preparation for high-resolution transmission electron microscopy (TEM) by employing epoxy to infiltrate into micropores. The epoxy can retain the agglomerate of cathode particles and avoid ion-beam-induced damage and contamination as well as suppress the side reactions with moisture and oxygen even after exposure to the air for a few months. The well-designed specimen preparation enables in-depth TEM investigation on the microstructure and phase transformation occurred severely at the outermost regions of porous cathode particles.

ОСОБЛИВОСТІ МОРФОЛОГІЧНИХ КОМПОНЕНТІВ ХРЯЩОВОГО ПОКРИТТЯ КОЛІННОГО СУГЛОБА НА МІКРОСТРУКТУРНОМУ ТА УЛЬТРАСТРУКТУРНОМУ РІВНЯХ НАПРИКІНЦІ ДВОХТИЖНЕВОЇ ВІДМІНИ ЕКСПЕРИМЕНТАЛЬНОГО ОПІОЇДНОГО ВПЛИВУ


 
 
 
 The work, presented below, compares pathomorphological changes of the articular cartilage in the distal epiphysis of the femur and proximal epiphyses of the tibia at the end of the 56th day in rats after two - week opioid withdrawal at the micro- and ultrastructural levels. The goal was achieved by micro- and ultrastructural visualization of the components of the articular cartilage. Specimen preparation for electron and light microscopy was done according to the generally accepted methods.The results of this study will form the basis for developing a comprehensive therapeutical approach in management of lesions of structural components of the articular cartilage in opioid chondrodystrophy.
 
 
 

Liftout of High-Quality Thin Sections of a Perovskite Oxide Thin Film Using a Xenon Plasma Focused Ion Beam Microscope.

It is shown that a xenon plasma focused ion beam (FIB) microscope is an excellent tool for high-quality preparation of functional oxide thin films for atomic resolution electron microscopy. Samples may be prepared rapidly, at least as fast as those prepared using conventional gallium FIB. Moreover, the surface quality after 2 kV final polishing with the Xe beam is exceptional with only about 3 nm of amorphized surface present. The sample quality was of a suitably high quality to allow atomic resolution high-angle annular dark field imaging and integrated differential phase contrast without any further preparation, and the resulting images were good enough for quantitative evaluation of atomic positions to reveal the oxygen octahedral tilt pattern. This suggests that such xenon plasma FIB instruments may find widespread application in transmission electron microscope and scanning transmission electron microscope specimen preparation.

Transmission Electron Microscopy Specimen Preparation for Two Dimensional Material Using Electron Beam Induced Deposition of a Protective Layer in the Focused Ion Beam Method

Transmission Electron Microscopy Specimen Preparation for Two Dimensional Material Using Electron Beam Induced Deposition of a Protective Layer in the Focused Ion Beam Method

Transmission Electron Microscope Specimen Preparation of Si-Based Anode Materials for Li-Ion Battery by Using Focused Ion Beam and Ultramicrotome

Transmission Electron Microscope Specimen Preparation of Si-Based Anode Materials for Li-Ion Battery by Using Focused Ion Beam and Ultramicrotome

Preparation of Tunable Microchips to Visualize Native Protein Complexes for Single-Particle Electron Microscopy.

Recent advances in technology have enabled single-particle electron microscopy (EM) to rapidly progress as a preferred tool to study protein assemblies. Newly developed materials and methods present viable alternatives to traditional EM specimen preparation. Improved lipid monolayer purification reagents offer considerable flexibility, while ultrathin silicon nitride films provide superior imaging properties to the structural study of protein complexes. Here, we describe the steps for combining monolayer purification with silicon nitride microchips to create a tunable approach for the EM community.

Transmission Electron Microscopy Specimen Preparation of Delicate Materials Using Tripod Polisher

Transmission electron microscopy (TEM) is a powerful tool for analyzing a broad range of materials and provides localized information about the microstructure. However, the analysis results are strongly influenced by the quality of the thin foil specimen. Sample preparation for TEM analysis requires considerable skill, especially when the area of interest is small or the material of interest is difficult to thin because of its high hardness and its mechanical instability when thinned. This article selectively reviews recent advances in TEM sample preparation techniques using a tripod polisher. In particular, it introduces two typical types (fl at type and wedge type) of TEM sample preparation and the benefits and drawbacks of each method; finally, a method of making better samples for TEM analysis is suggested.

Qualifying Electrically Conductive Cold Embedding-Media for Scanning Electron Microscopy

The choice of embedding-media for metallographic specimen preparation, where subsequent scanning electron microscopy will be performed, is highly important since the quality of the analytical results in the specimen’s near-surface region is limited by the properties of the embedding-material. Due to their high electrical conductivity and only slight tendency to form gaps between near the sample surface, warm curing embedding-media are usually best suited. Hot embedding-presses use relatively high pressures at elevated temperatures that can be detrimental for sensitive materials or coatings. Cold embedding with conventional embedding-media, which does not require high pressure mounting, requires compromising for a material with a lower electric conductivity, a higher amount of pores in the material, and inferior interfacial connectivity to the specimen. For this reason, the development of alternative electrically conductive and cold hardening embedding-media is of great interest for metallographic sample preparation of electron microscopy specimens. In the present study, various embedding-media were evaluated with respect to their suitability for scanning electron microscopy and a novel conductive mounting compound has been identified and qualified.

Parallel preparation of plan-view transmission electron microscopy specimens by vapor-phase etching with integrated etch stops

Specimen preparation remains a practical challenge in transmission electron microscopy and frequently limits the quality of structural and chemical characterization data obtained. Prevailing methods for thinning of specimens to electron transparency are serial in nature, time consuming, and prone to producing artifacts and specimen failure. This work presents an alternative method for the preparation of plan-view specimens using isotropic vapor-phase etching with integrated etch stops. An ultrathin amorphous etch-stop layer simultaneously serves as an electron transparent support membrane whose thickness is defined by a controlled growth process such as atomic layer deposition with sub-nanometer precision. This approach eliminates the need for mechanical polishing or ion milling to achieve electron transparency, and reduces the occurrence of preparation induced artifacts. Furthermore, multiple specimens from a plurality of samples can be thinned in parallel due to high selectivity of the vapor-phase etching process. These features enable dramatic reductions in preparation time and cost without sacrificing specimen quality and provide advantages over wet etching techniques. Finally, we demonstrate a platform for high-throughput transmission electron microscopy of plan-view specimens by combining the parallel preparation capabilities of vapor-phase etching with wafer-scale micro- and nanofabrication.