Electron Microscopy Sample Preparation Research Articles

Metrology of Sample Preparation for Electron Microscopy: Application to Strain Measurements

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How innovations in methodology offer new prospects for volume electron microscopy

Detailed knowledge of biological structure has been key in understanding biology at several levels of organisation, from organs to cells and proteins. Volume electron microscopy (volume EM) provides high resolution 3D structural information about tissues on the nanometre scale. However, the throughput rate of conventional electron microscopes has limited the volume size and number of samples that can be imaged. Recent improvements in methodology are currently driving a revolution in volume EM, making possible the structural imaging of whole organs and small organisms. In turn, these recent developments in image acquisition have created or stressed bottlenecks in other parts of the pipeline, like sample preparation, image analysis and data management. While the progress in image analysis is stunning due to the advent of automatic segmentation and server‐based annotation tools, several challenges remain. Here we discuss recent trends in volume EM, emerging methods for increasing throughput and implications for sample preparation, image analysis and data management.

Site-specific preparation of plan-view samples with large field of view for atomic resolution STEM and TEM studies of rapidly solidified multi-phase Al Cu thin films

The present work describes application of a method for site-specific plan-view sample preparation for atomic column resolution scanning transmission and transmission electron microscopy (STEM and TEM) from free-standing thin films of multi-phase Al-alloys after laser irradiation induced rapid solidification (RS). The electron-transparent multilayer thin film samples (amorphous Si3N4 substrate plus metal alloy layer) had a total initial thickness of ≈ 130–200 nm. At such large total sample thickness frequently multiple grains of the same or different phases overlap along the electron beam path in the RS microstructures of the alloys. Consequently, accurate atomic resolution images in TEM and STEM mode, and composition sensitive analytical data of the metastable microstructural features presenting in the RS microstructures cannot be obtained with confidence. Using low-energy concentrated Ar ion beam milling, locally thinned regions with thickness of 20–30 nm with large fields of view ≥ (30 μm)2 suitable for high-resolution TEM/STEM studies have been created with micron-scale site-specificity. As example application, atomic scale resolution TEM/STEM imaging has been performed of the banded grain regions of the RS microstructure established in multi-phase AlCu alloy thin films. The sample preparation strategy described here appears to be readily applicable to freestanding thin film specimens in general.

Exploring Charged Defects in Ferroelectrics by the Switching Spectroscopy Piezoresponse Force Microscopy.

Monitoring the charged defect concentration at the nanoscale is of critical importance for both the fundamental science and applications of ferroelectrics. However, up-to-date, high-resolution study methods for the investigation of structural defects, such as transmission electron microscopy, X-ray tomography, etc., are expensive and demand complicated sample preparation. With an example of the lanthanum-doped bismuth ferrite ceramics, a novel method is proposed based on the switching spectroscopy piezoresponse force microscopy (SSPFM) that allows probing the electric potential from buried subsurface charged defects in the ferroelectric materials with a nanometer-scale spatial resolution. When compared with the composition-sensitive methods, such as neutron diffraction, X-ray photoelectron spectroscopy, and local time-of-flight secondary ion mass spectrometry, the SSPFM sensitivity to the variation of the electric potential from the charged defects is shown to be equivalent to less than 0.3 at% of the defect concentration. Additionally, the possibility to locally evaluate dynamics of the polarization screening caused by the charged defects is demonstrated, which is of significant interest for further understanding defect-mediated processes in ferroelectrics.

Living up to its potential—Direct-write nanofabrication with focused electron beams

Over its rather long history, focused electron beam induced deposition (FEBID) has mostly been used as an auxiliary process in passivating surfaces in sample preparation for transmission electron microscopy. This has changed over the last one and a half decades. On the one hand, FEBID has been established as the leading technical approach to lithography mask repair on the industrial scale. On the other hand, FEBID-related technical and methodological developments, FEBID-derived materials, and FEBID-based device fabrication have had a significant impact in various areas of basic and applied research, such as nanomagnetism and superconductivity, plasmonics, and sensing. Despite this dynamic development, the FEBID user base does still form a rather exclusive club of enthusiasts. In this Perspective, our aim is to provide sufficient insight into the basics of FEBID, its potential, as well as its challenges, to scientists working in the broader fields of materials science, nanotechnology, and device development. It is our hope to spark growing interest and even excitement into FEBID which, as we believe, still has to live up to its full potential.

Understanding the formation of antiphase boundaries in layered oxide cathode materials and their evolution upon electrochemical cycling

Understanding the formation of antiphase boundaries in layered oxide cathode materials and their evolution upon electrochemical cycling

Scanning Electron Microscope Observation of Arabidopsis Pollen Under Two Treatment Conditions

We observed pollen in the late development stages of fresh wild Arabidopsis thaliana by scanning electron microscope, using direct spraying of gold and the conventional biological electron microscope sample preparation method, and made comparative analyses. Arabidopsis pollen prepared according to the conventional biological electron microscope sample preparation method can maintain intact and clear outer wall structure under scanning electron microscopy. However, after direct spraying of gold, only mature pollen could be observed relatively clear outer wall structure, and no clear observation results could be obtained for pollen in other stages. Our results showed that the outer wall morphology of the mature pollen can be observed by direct gold spraying with fresh materials. This method is easy to operate and saves time. For the immature pollen, especially the pollen in the later stage of development, direct gold spraying will affect the pollen morphology, so we recommend using the conventional biological electron microscope sample preparation method to obtain the best shooting effect. The results of this study might provide some useful reference for researchers to observe the pollen outer wall structure in the late development stages of Arabidopsis thaliana by scanning electron microscope.

Expanding the Capability of Xe Plasma Focused Ion Beam Sample Preparation for Transmission Electron Microscopy

Expanding the Capability of Xe Plasma Focused Ion Beam Sample Preparation for Transmission Electron Microscopy

Use of cleaved wedge geometry for plan‐view transmission electron microscopy sample preparation

A fast, convenient, and easy to perform method for preparing plan-view transmission electron microscopy (TEM) specimens of brittle materials is proposed. The method is ideal for thin films/coatings and based on obtaining wedge-shape geometries of the samples via conventional cutting and cleaving followed by gentle focused ion beam (FIB) milling to electron transparency. It enables multiple parallel windows for depth sectioning of the samples and facilitates FIB lift-out procedure. The method has been successfully applied for preparing high-quality plan-view TEM samples for a range of films deposited on Si, SiC, and Al2 O3 which significantly enhances throughput and reduces time at the FIB. The method further offers high success rate even for the novice, stable handling and reproducibility, which greatly widens the application of advanced plan-view TEM studies in material science.

Antibacterial Effect of Endodontic Disinfections on Enterococcus Faecalis in Dental Root Canals-An In-Vitro Model Study.

Enterococcus faecalis (E. faecalis) is rather unsusceptible to many root canal disinfections which often cause a therapeutic problem. Therefore, the present in vitro study observed the efficiency of different endodontic antiseptics in their capability to suppress E. faecalis, especially inside dentinal tubules. Prior to any testing, root canals of extracted third human molars were inoculated with E. faecalis for 48 h. Antiseptic dressings with chloramine-T or calcium hydroxide (CaOH) for 24 h or irrigations with 1.3% sodium hypochlorite (NaOCl) were applied with n = 10 in each group. As control irrigation with normal saline was used. All treated canals were manually enlarged from size ISO 50 to 110 and the ablated dentin debris was subjected to microbial culture analysis. Bacterial colonization of the dentinal tubules up to 300 µm was verified by scanning electron microscopy and histological sample preparation. Application of crystalline chloramine-T caused total bacterial suppression inside the dentinal tubules. Dressings with CaOH showed only minor effects. Irrigation with NaOCl caused total eradication of bacteria adhering to the root canal walls, but also failed to completely suppress E. faecalis inside the dentinal tubules. The study showed that chloramine-T is of strong antiseptic activity and also efficient in suppressing E. faecalis inside dentinal tubules.

Transmission electron microscopy sample preparation method for micrometer-sized powder particles using focused ion beam

A TEM sample preparation technique for micrometer-sized powder particles in the 1−10 μm size range is proposed, using a focused ion beam (FIB) system. It is useful for characterizing elemental distributions across an entire cross-section of a particle. It is a simple and universal method without using any embedding agent, enabling the powder particles with different size, shape or orientation to be easily selected based on the SEM observations. The suitable particle is covered with Pt coating layers through an ion-beam-assisted deposition. The Pt coating layers provide sufficient support for the TEM lamella. A small piece of tungsten needle is used as a support under the particle by taking a series of operations using a micromanipulator. The particle can be precisely thinned by the ion beam to be suitable for both TEM observation and EDX elemental mapping. This novel technique reduces the TEM sample preparation time to a few hours, allowing much higher efficiency compared to complicated and time-consuming embedding methods.

Comparing Xe+ pFIB and Ga+ FIB for TEM sample preparation of Al alloys: Minimising FIB-induced artefacts.

Recently, the dual beam Xe+ plasma focused ion beam (Xe+pFIB) instrument has attracted increasing interest for site‐specific transmission electron microscopy (TEM) sample preparation for a local region of interest as it shows several potential benefits compared to conventional Ga+FIB milling. Nevertheless, challenges and questions remain especially in terms of FIB‐induced artefacts, which hinder reliable S/TEM microstructural and compositional analysis. Here we examine the efficacy of using Xe+ pFIB as compared with conventional Ga+ FIB for TEM sample preparation of Al alloys. Three potential source of specimen preparation artefacts were examined, namely: (1) implantation‐induced defects such as amophisation, dislocations, or ‘bubble’ formation in the near‐surface region resulting from ion bombardment of the sample by the incident beam; (2) compositional artefacts due to implantation of the source ions and (3) material redeposition due to the milling process. It is shown that Xe+pFIB milling is able to produce improved STEM/TEM samples compared to those produced by Ga+ milling, and is therefore the preferred specimen preparation route. Strategies for minimising the artefacts induced by Xe+pFIB and Ga+FIB are also proposed.Lay Description FIB (focused ion beam) instruments have become one of the most important systems in the preparation of site‐specific TEM specimens, which are typically 50‐100 nm in thickness. TEM specimen preparation of Al alloys is particularly challenging, as convention Ga‐ion FIB produces artefacts in these materials that make microstructural analysis difficult or impossible. Recently, the use of noble gas ion sources, such as Xe, has markedly improved milling speeds and is being used for the preparation of various materials. Hence, it is necessary to investigate the structural defects formed during FIB milling and assess the ion‐induced chemical contamination in these TEM samples. Here we explore the feasibility and efficiency of using Xe+PFIB as a TEM sample preparation route for Al alloys in comparison with the conventional Ga+FIB.

Correlative super-resolution fluorescence and electron cryo-microscopy based on cryo-SOFI.

The combination of super-resolution fluorescence microscopy and electron microscopy at ambient temperatures has become an established technique and a broad variety of modalities are now available to the cell biology community. In contrast, correlative cryogenic super-resolution fluorescence and electron microscopy (super-resolution cryo-CLEM) is just emerging. Aside from technical challenges, one of the major issues is the risk of devitrification of the specimen caused by the laser intensities required for super-resolution imaging. Cryo-SOFI (cryogenic super-resolution optical fluctuation imaging) allows the reconstruction of super-resolution images at particularly low laser intensities. It is fully compatible with the standard sample preparation for cryogenic electron microscopy (cryo-EM) and fairly easy to implement in any standard cryogenic fluorescence microscope.

Laser ablation sample preparation for atom probe tomography and transmission electron microscopy

Laser ablation is capable of removing large volumes of material with micron scale precision at very high speeds. This makes it an ideal tool for the initial stage of preparation of samples for atom probe and electron microscopy studies. However, the thermal nature of the laser ablation process is such that thermal and mechanical damage is induced in the samples in the form of zones of recrystallisation and stress induced deformation. For the analysis of nanometer-sized samples, such as those required for atom probe tomography and transmission electron microscopy, it is necessary to ensure that any damage induced during sample preparation will not introduce artefacts and that specimens are representative of the microstructure of the bulk sample. Here we have undertaken an analysis of the damage caused during sample preparation through a study of pure aluminium and phosphorous doped silicon wafers. Our findings indicate that recrystallisation and stress induced misorientations occur in pure aluminium at the micron scale, however, no detectable damage is observed in the silicon sample.

Anomalous enhancement of focused ion beam etching by single raster propagating toward ion beam at glancing incidence

Focused ion beam (FIB) sample preparation for electron microscopy often requires large volumes of materials to be removed. Prior efforts to increase the rate of bulk material removal were mainly focused on increasing the primary ion beam current. Enhanced yield of etching at glancing ion beam incidence is known but has not found widespread use in practical applications. In this study, etching at glancing ion beam incidence was explored for its advantages in increasing the rate of bulk material removal. Anomalous enhancement of material removal at glancing angles of ion beam incidence was observed with single-raster etching in along-the-slope direction with toward-FIB direction of raster propagation. Material removal was inhibited in an away-from-FIB direction of raster propagation. The effects of glancing angles and ion doses on depth of cut and volume of removed materials were also recorded. We demonstrated that the combination of single-raster FIB etching at glancing incidence in along-the-slope direction with toward-FIB raster propagation and a “staircase” type of etching strategy holds promise for reducing the processing time for bulk material removal in FIB sample preparation applications.

Ga-contamination-free scanning transmission electron microscope sample preparation by rectangular-shaped oxygen-ion-beam thinning using projection ion beam optical system

Rectangular-shaped oxygen O2+ ion beam thinning for scanning transmission electron microscope (STEM) sample preparation was investigated using a projection ion beam optical system equipped with a duoplasmatron gas ion source. The ion current can be increased by increasing the area of the rectangular-shaped oxygen ion beam and thereby overcome the low brightness of the ion source. Rectangular-shaped O2+ ion beams with different amounts of projection beam edge blur were formed in the X and Y directions by using a limiting mask between the ion source and projection mask. Gallium-contamination-free STEM sample preparation (rough, medium, and fine milling) was demonstrated using a projection ion beam optical system and three types of rectangular-shaped oxygen ion beams.

Electron Microscopy Views of Dimorphic Chloroplasts in C4 Plants.

C4 plants enhance photosynthesis efficiency by concentrating CO2 to the site of Rubisco action. Chloroplasts in C4 plants exhibit structural dimorphism because thylakoid architectures vary depending on energy requirements. Advances in electron microscopy imaging capacity and sample preparation technologies allowed characterization of thylakoid structures and their macromolecular arrangements with unprecedented precision mostly in C3 plants. The thylakoid is assembled during chloroplast biogenesis through collaboration between the plastid and nuclear genomes. Recently, the membrane dynamics involved in the assembly process has been investigated with 3D electron microscopy, and molecular factors required for thylakoid construction have been characterized. The two classes of chloroplasts in C4 plants arise from common precursors, but little is known about how a single type of chloroplasts grow, divide, and differentiate to mature into distinct chloroplasts. Here, we outline the thylakoid structure and its assembly processes in C3 plants to discuss ultrastructural analyses of dimorphic chloroplast biogenesis in C4 plant species. Future directions for electron microscopy research of C4 photosynthetic systems are also proposed.

Cryo-Electron microscopy for the study of self-assembled poly(ionic liquid) nanoparticles and protein supramolecular structures

Cryo-electron microscopy (cryo-EM) is a powerful structure determination technique that is well-suited to the study of protein and polymer self-assembly in solution. In contrast to conventional transmission electron microscopy (TEM) sample preparation, which often times involves drying and staining, the frozen-hydrated sample preparation allows the specimens to be kept and imaged in a state closest to their native one. Here, we give a short overview of the basic principles of Cryo-EM and review our results on applying it to the study of different protein and polymer self-assembled nanostructures. More specifically, we show how we have applied cryo-electron tomography (cryo-ET) to visualize the internal morphology of self-assembled poly(ionic liquid) nanoparticles and cryo-EM single particle analysis (SPA) to determine the three-dimensional (3D) structures of artificial protein microtubules.Grapical abstract

Progress of Sample Preparation for Electron Microscopy Using Ion Beam

Progress of Sample Preparation for Electron Microscopy Using Ion Beam

Features of large-scale thin foils fabrication for transmission electron microscopy by focused ion beam

Focused ion beam (FIB) sample preparation for transmission electron microscopy is a well established technique. However there are some problems related with high-quality large-scale (over 10 um) sample preparation of thin foils. In this work we demonstrate the successful preparation of such samples on example of ferrite steel sample and present the refined lift-out preparation technique, which was applied here. All sample preparation has been carried out in conventional single-beam FIB Hitachi FB-2100 with additional low-kV Ar+ ion polishing.