Electron Microscopy Sample Preparation Research Articles

Serial block-face scanning electron microscopy of adherent cells on thin plastic substrate

Abstract Serial block-face (SBF) scanning electron microscopy (SEM) is used for imaging the entire internal ultrastructure of cells, tissue samples or small organisms. Here, we present a workflow for SBF SEM of adherent cells, such as Giardia parasites and HeLa cells, attached to the surface of a plastic culture dish, which preserves the interface between cells and plastic substrate. Cells were embedded in situ on their substrate using silicone microwells and were mounted for cross-sectioning which allowed SBF imaging of large volumes and many cells. A standard sample preparation and embedding protocol for thin section electron microscopy provided already sufficient resolution and image quality to visualize larger structures. To improve resolution and image quality of SBF imaging, we stepwise tested modifications of the protocol, such as the moderate increase of the heavy metal content of the sample. Modifications of the embedding by either the reduction of the resin layer (minimal embedding) or the addition of silver colloid to the resin were evaluated at high and low vacuum imaging conditions. The optimized sample preparation protocol is very similar to the standard preparation protocol for thin section electron microscopy, so that the samples can also be used for this application. The protocol applies a higher concentration of osmium tetroxide, a higher temperature for heavy metal incubation and an additional lead en bloc staining. In summary, the presented workflow provides a generic and adaptable solution for studying adherent cells by SBF SEM.

Studying crystallisation processes using electron microscopy: The importance of sample preparation.

We present a comparison of common electron microscopy sample preparation methods for studying crystallisation processes from solution using both scanning and transmission electron microscopy (SEM and TEM). We focus on two widely studied inorganic systems: calcium sulphate, gypsum (CaSO4·2H2O) and calcium carbonate (CaCO3). We find significant differences in crystallisation kinetics and polymorph selection between the different sample preparation methods, which indicate that drying and chemical quenching can induce severe artefacts that are capable of masking the true native state of the crystallising solution. Overall, these results highlight the importance of cryogenic (cryo)-quenching crystallising solutions and the use of full cryo-TEM as the most reliable method for studying the early stages of crystallisation.

Enabling focused ion beam sample preparation for application in reverse tip sample scanning probe microscopy

Focused ion beam (FIB) has become a powerful tool for transmission electron microscopy sample preparation in the nanoelectronics industry and has in recent years also shown its benefits for specific preparation steps in electrical scanning probe microscopy (SPM). Most recently, a novel SPM approach – so-called reverse tip sample (RTS) SPM – has been proposed in which the position of sample and tip are switched compared to standard SPM; in RTS SPM the sample is attached to the end of a cantilever beam. To achieve this configuration, the region of interest must first be extracted from a substrate and then needs to be reliably fixed to the cantilever by FIB. Therefore, we have explored and developed dedicated FIB preparation methods for RTS SPM in this work. Our established procedures ensure a strong mechanical and good electrical connection of the sample to the cantilever for both cross-section and top view sample preparation. Furthermore, we introduce an approach for mounting samples from a full wafer size workflow. This paper presents the developed FIB procedures and discusses the quality and stability of all mounted samples and their electrical evaluation in RTS SPM.

Rapid in-EPON CLEM: Combining fast and efficient labeling of self-labeling enzyme tags with EM-resistant Janelia Fluor dyes and StayGold

Correlative light and electron microscopy (CLEM) combines light microscopy (LM) of fluorescent samples to ultrastructural analyses by electron microscopy (EM). Pre-embedding CLEM often suffers from inaccurate correlation between LM and EM modalities. Post-embedding CLEM enables precise registration of structures directly on EM sections, but requires fluorescent markers withstanding EM sample preparation, especially osmium tetroxide fixation, dehydration and EPON embedding. Most fluorescent proteins (FPs) lose their fluorescence during such conventional embedding (CE), but synthetic dyes represent promising alternatives as their stability exceeds those of FP. We analyzed various Janelia Fluor dyes and TMR conjugated to ligands for self-labeling enzymes, such as HaloTag, for fluorescence preservation after CE. We show that TMR, JF525, JF549, JFX549 and JFX554 retain fluorescence, with JFX549 and JFX554 yielding best results overall, also allowing integration of high-pressure freezing and freeze substitution. Furthermore, we found the recently published FP StayGold to resist CE, facilitating dual-fluorescence in-resin CLEM.

An evaluation of Ar ion milling in TEM sample preparation by energy-filtered TEM technique

Irradiated materials impose unique challenges when it comes to sample preparation for transmission electron microscopy (TEM) analysis. After the preparation of TEM samples by electrolytic polish, Ar ion milling was usually used to remove the surface contamination, as well as to thin the samples to desired thickness. Therefore, it is necessary to evaluate the advantages and limitations of Ar ion milling technique. In this work, the optimum milling condition with minimal surface artifacts was determined by Ar ion milling with various incident angles and beam energies, and the high-resolution TEM (HR-TEM) examinations confirmed the optimum condition to be 4.0keV Ar ions with incident angles of ±4°. In addition, Ar ion milling and subsequent thickness measurement by energy-filtered TEM (EF-TEM) were conducted to determine the thinning rate. It is found that electrolytic polished TEM lamella has a wedge-shape structure with a very sharp edge, and the thinning rates varied from 9.7±1.4nm/minute to 5.0±2.3nm/minute across the TEM lamella for 4.0keV Ar ions. However, the thinning rates for 2keV and 1keV Ar ions were under the examination limit, and they were supposed not sufficient to thin the tungsten samples. Even though the exact thinning rate varies for different materials, the general trends for the Ar ion milling observed in this study can be used as a guidance.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) culture and sample preparation for correlative light electron microscopy.

Correlative Light Electron Microscopy (CLEM) is a powerful technique to investigate the ultrastructure of specific cells and organelles at sub-cellular resolution. Transmission Electron Microscopy (TEM) is particularly useful to the field of virology, given the small size of the virion, which is below the limit of detection by light microscopy. Furthermore, viral infection results in the rearrangement of host organelles to form spatially defined compartments that facilitate the replication of viruses. With the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there has been great interest to study the viral replication complex using CLEM. In this chapter we provide an exemplary workflow describing the safe preparation and processing of cells grown on coverslips and infected with SARS-CoV-2.

Determining Macromolecular Structures Using Cryo-Electron Microscopy.

Structural insights into macromolecular and protein complexes provide key clues about the molecular basis of the function. Cryogenic electron microscopy (cryo-EM) has emerged as a powerful structural biology method for studying protein and macromolecular structures at high resolution in both native andnear-native states. Despite the ability to get detailed structural insights into the processes underlying protein function using cryo-EM, there has been hesitancy amongst plant biologists to apply the method for biomolecular interaction studies. This is largely evident from the relatively fewer structural depositions of proteins and protein complexes from plant origin in electron microscopy databank. Even though the progress has been slow, cryo-EM has significantly contributed to our understanding of the molecular biology processes underlying photosynthesis, energy transfer in plants, besides viruses infecting plants. This chapter introduces sample preparation for both negative-staining electron microscopy (NSEM) and cryo-EM for plant proteins and macromolecular complexes and data analysis using single particle analysis for beginners.

Sample preparation and data collection for serial block face scanning electron microscopy of mammalian cell monolayers.

Volume electron microscopy encompasses a set of electron microscopy techniques that can be used to examine the ultrastructure of biological tissues and cells in three dimensions. Two block face techniques, focused ion beam scanning electron microscopy (FIB-SEM) and serial block face scanning electron microscopy (SBF-SEM) have often been used to study biological tissue samples. More recently, these techniques have been adapted to in vitro tissue culture samples. Here we describe step-by-step protocols for two sample embedding methods for in vitro tissue culture cells intended to be studied using SBF-SEM. The first focuses on cell pellet embedding and the second on en face embedding. En face embedding can be combined with light microscopy, and this CLEM workflow can be used to identify specific biological events by light microscopy, which can then be imaged using SBF-SEM. We systematically outline the steps necessary to fix, stain, embed and image adherent tissue culture cell monolayers by SBF-SEM. In addition to sample preparation, we discuss optimization of parameters for data collection. We highlight the challenges and key steps of sample preparation, and the consideration of imaging variables.

Effect of electron microscopy sample preparation protocol on the preservation of liposomes in cell culture

Effect of electron microscopy sample preparation protocol on the preservation of liposomes in cell culture

A Comprehensive Approach to Sample Preparation for Electron Microscopy and the Assessment of Mitochondrial Morphology in Tissue and Cultured Cells (Adv. Biology 10/2023)

A Comprehensive Approach to Sample Preparation for Electron Microscopy and the Assessment of Mitochondrial Morphology in Tissue and Cultured Cells (Adv. Biology 10/2023)

Preparing metallic bulk samples for transmission electron microscopy analysis via laser ablation in an acidic liquid

The laser ablation process has been presented as a state-of-the-art and robust method for eliminating surface layers of metallic bulk materials on the micro/nanoscale with considerable accuracy and speed. These capabilities make it an ideal instrument for the primary step of metallic bulk sample preparation for transmission electron microscopy (TEM) analysis with minimal damage to the crystal structure of the sample. This work prepared a pure aluminum plate by fiber laser scanning immersed in a 5 % perchloric acid and 95 % ethanol (by volume) solution. The laser ablation in an acidic liquid condition was used in the chemical etching process, to prevent oxidation, minimize the heat-affected region, and induce crystalline defects. For this purpose, a continuous wave fiber laser with a power of 100 W, a wavelength of 1064 nm, and a focusing distance of 305 mm was applied. The TEM analysis for the as-prepared sample by the laser ablation process was compared to the standard jet electro-polishing method in the same solution. The results showed that crystal structure variations such as grain size and lattice defects did not occur in the aluminum sample due to the thermal effects of the laser ablation process. The average size of the grains and zone axis were about 600–800 nm and z = [011], respectively, in as-prepared samples by both methods; In addition, very few dislocations were observed in the grain boundaries and inside the grains. Therefore, based on the obtained results, laser ablation could be considered an eco-friendly, comfortable, fast, and cost-effective method with a small volume of chemicals used, compared to the standard jet electro-polishing method for the TEM preparation of the metallic bulk samples.

Ion beam processing with an ultra-low energy Ar+ micro-polisher: From fundamental understanding to process optimisation

Ion beam processes are used in many applications, including surface patterning, milling, implantation, sample characterisation and sample preparation for electron microscopy. The use of ultra-low energies in the sub-keV range allows to reduce the implantation depth of the ion species, and hence to reduce the irradiation induced damage in the sample. In the current study we explore the potential of a simple experimental setup for ultra-low energy ion beam processes in the impact energy range of 50 eV to 500 eV and pay attention to the ion-beam induced processes on the sample surface. Our work shows that when ion beam process related to milling need to be well controlled, ion beam induced deposition of residual gas molecules in the instrument chamber needs to be considered. Depending on ion energy and irradiation density, ion induced sputtering and deposition can compete. Hence, the quality of the vacuum can significantly impact the process. Experimental sputtering yields were difficult to obtain, therefore reference values for a large range of angles and energies were simulated.

Flash electropolishing of BCC Fe and Fe-based alloys

The preparation of transmission electron microscopy (TEM) samples is a critical step in the characterization of materials, and the focused ion beam (FIB) technique is a commonly used method. However, a significant limitation of this technique is the FIB-induced damages on the foil surfaces, which can obscure the real features of interest, particularly in radiation effects studies. To overcome this limitation, this study presents a detailed description of the flash electropolishing technique, which can be used to remove the FIB damage from samples. The flash electropolishing technique has been successfully applied to a range of materials, including Fe, Fe-based model alloys, commercial Fe-Cr alloys, and advanced Fe-Cr alloys, both in their as-received and ion-irradiated states. Furthermore, the parameters used for Fe-Cr model alloys can be adjusted for commercial and advanced alloys with minimal modifications. The study also examined the effects of electropolishing variables, such as perchloric acid concentration, electropolishing temperature, Cr concentration, and voltage. Qualitatively, a general trend and scoping test strategy is explored in our experiments. Overall, the flash electropolishing technique offers a promising solution to the challenges posed by FIB-induced damages in the preparation of TEM samples.

Stability of the Interface Between LIPON and LCO During TEM Sample Preparation by FIB.

An amorphous interphase between lithium phosphorus oxynitride (LIPON) solid electrolyte and lithium cobalt oxide (LCO) has been reported recently in several electron microscopy studies of Li ion thin-film micro-batteries (TFMB), along with its implications to battery operation. However, the origin of the observed interphase at the as-made LIPON/LCO interface remained obscure. In this work, this interface has been characterized comprehensively by scanning electron microscope (SEM) imaging at all steps of the in situ focused ion beam (FIB) lift-out procedure for transmission electron microscopy (TEM) sample preparation. It was found that the interphase is formed during TEM lamella preparation when the portion of LIPON layer contained within the lamella is physically disconnected from the rest of the LIPON layer by FIB. Therefore, it was demonstrated that a disordered interphase can form in LCO at its interface with LIPON during TEM sample preparation by the FIB lift-out procedure and that subtle nature of the interphase formation makes it likely to go unnoticed during the preparation. This interphase was not produced even after galvanostatic charging of a battery with a Li metal anode but inevitably appeared after the FIB lift-out of that sample.

A Comprehensive Approach to Sample Preparation for Electron Microscopy and the Assessment of Mitochondrial Morphology in Tissue and Cultured Cells.

Mitochondria respond to metabolic demands of the cell and to incremental damage, in part, through dynamic structural changes that include fission (fragmentation), fusion (merging of distinct mitochondria), autophagic degradation (mitophagy), and biogenic interactions with the endoplasmic reticulum (ER). High resolution study of mitochondrial structural and functional relationships requires rapid preservation of specimens to reduce technical artifacts coupled with quantitative assessment of mitochondrial architecture. A practical approach for assessing mitochondrial fine structure using two dimensional and three dimensional high-resolution electron microscopy is presented, and a systematic approach to measure mitochondrial architecture, including volume, length, hyperbranching, cristae morphology, and the number and extent of interaction with the ER is described. These methods areused to assess mitochondrial architecture in cells and tissue with high energy demand, including skeletal muscle cells, mouse brain tissue, and Drosophila muscles. The accuracy of assessment isvalidated in cells and tissue with deletion of genes involved in mitochondrial dynamics.

Express-diagnosis of the diseases of the eye and its appendages by scanning electron microscopy with supravital staining

This review describes the history of development of a new line of chemical reagents that prompts to significantly reevaluate the application of scanning electron microscopy (SEM) in medical and biological studies, particularly in ophthalmology; considers the establishing of SEM as an analytical method; covers the problems in its application associated with the needs of clinical medicine and the complexities of biological sample preparation for electron microscopy. The article also presents in chronological order the technical solutions associated with creating a unique line of reagents for supravital staining. The multitude of technical solutions allows considering SEM as a method of express diagnostics. The review discusses examples of practical application of these methods for solving certain cases in clinical ophthalmology. The niche of SEM is considered among other methods of clinical diagnostics, as well as its future development involving the use of artificial intelligence.

Exploring the influence of focused ion beam processing and scanning electron microscopy imaging on solid-state electrolytes.

Performing reliable preparation of transmission electron microscopy (TEM) samples is the necessary basis for a meaningful investigation by ex situ and even more so by in situ TEM techniques, but it is challenging using materials that are sensitive to electron beam irradiation. Focused ion beam is currently the most commonly employed technique for a targeted preparation, but the structural modifications induced during focused ion beam preparation are not fully understood for a number of materials. Here, we have investigated the impact of both the electron and the Ga+ ion beam on insulating solid-state electrolytes (lithium phosphorus oxynitride, Na-β"-alumina solid electrolyte and Na3.4Si2.4Zr2P0.6O12 (NaSICON)) and observed significant lithium/sodium whisker growth induced by both the electron and ion beam already at fairly low dose, leading to a significant change in the chemical composition. The metal whisker growth is presumably mainly due to surface charging, which can be reduced by coating with a gold layer or preparation under cryogenic conditions as efficient approaches to stabilize the solid electrolyte for scanning electron microscopy imaging and TEM sample preparation. Details on the different preparation approaches, the acceleration voltage dependence and the induced chemical and morphological changes are reported.

Ga+ Focused Ion Beam Damage in n-type Ga2O3 and Its Recovery after Annealing Treatment

In this study, the focused ion beam (FIB) Ga+ irradiation damage on β-Ga2O3 Schottky diode structure under different ion energies were tested, and the effective recovery of diode characteristics after rapid thermal annealing (RTA) post-annealing were also studied.β-Ga2O3 based Schottky diodes have attracted increasing attention because of the prospects for use in next-generation high-power electronics. Focused ion beam (FIB) machining is one of the newest processing techniques, which is mainly used in semiconductor and chip manufacture and transmission electron microscopy (TEM) sample preparation. During FIB sample preparation, the energetic Ga ions are incident onto the sample to induce physical sputtering of the material, which in turn, modifies its chemical composition, crystallinity and electrical properties. It has been found in many studies that 20-30 nm amorphous layer can form on silicon after irradiation with 30 keV Ga ions. From our previous study, the FIB ion beam induced damages could cause significant degradation on diode on-resistance and turn-on voltage. Milling at lower energy has been used to reduce the electrical damage. However low energy milling is more time consuming and less precise. Short-time, high-temperature post-annealing treatments, such as RTA has been proved could remove the crystal damage. Thus, there is a great need to determine the maximal ion beam energy and post-annealing temperature for FIB without degrading diode characteristics.In this work, energies ranging from 2 keV to 30 keV were employed to mill off a part of Ga2O3 epi-layer and Schottky diodes were fabricated on milled surface. Forward IV characteristic shows less degradations with lower ion beam energies. For the diodes exposed to 2, 5, and 10 keV Ga+ ion beams exhibited significant recovery of diode on resistance and turn-on voltage after 300°C RTA annealing. For the diodes exposed to 30 keV Ga+ ion beams recovered significantly after 400 °C annealing. In conclusion, decreasing the FIB energy shows effective alleviations on the FIB damage, and post annealing treatment effectively recovered the damage from Ga+ implantation.This is the first time the thermal annealing treatment was performed for Ga2O3 Schottky diode recovering from FIB cutting, it is an effective and simple way to mitigate the degradation, especially for higher ion beam energies. This work could broaden the FIB’s usability and release tremendous potential as a specimen preparation and imaging tool in materials science applications. Figure 1

Influence of water contamination on the sputtering of silicon with low-energy argon ions investigated by molecular dynamics simulations.

Focused ion beams (FIB) are a common tool in nanotechnology for surface analysis, sample preparation for electron microscopy and atom probe tomography, surface patterning, nanolithography, nanomachining, and nanoprinting. For many of these applications, a precise control of ion-beam-induced processes is essential. The effect of contaminations on these processes has not been thoroughly explored but can often be substantial, especially for ultralow impact energies in the sub-keV range. In this paper we investigate by molecular dynamics (MD) simulations how one of the most commonly found residual contaminations in vacuum chambers (i.e., water adsorbed on a silicon surface) influences sputtering by 100 eV argon ions. The incidence angle was changed from normal incidence to close to grazing incidence. For the simulation conditions used in this work, the adsorption of water favours the formation of defects in silicon by mixing hydrogen and oxygen atoms into the substrate. The sputtering yield of silicon is not significantly changed by the contamination, but the fraction of hydrogen and oxygen atoms that is sputtered largely depends on the incidence angle. This fraction is the largest for incidence angles between 70 and 80° defined with respect to the sample surface. Overall, it changes from 25% to 65%.

A precision core drill for transmission electron microscopy sample preparation produced by 3D printing

A precision core-drilling machine has been designed and constructed almost entirely from 3D printing. The purpose of the drill is to extract 3 mm diameter discs from thin slices of mainly hard brittle materials for analysis in a transmission electron microscope. Novel methods of producing a slide bearing and shaft couplings in 3D printed components have been developed. Design principles and material selection are discussed, and the drill is evaluated by preparing samples from silicon and aluminium. The device can be self-made by any laboratory with a 3D printer, can be recycled when no longer required and can help labs reach their sustainable development goals.