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.

A scalable, passive sampling method for the quantification of airborne allergens

Traditionally, airborne concentrations of aeroallergens are sampled in a single location by an active sampler, which requires electricity and regular maintenance. However, aeroallergen concentrations may vary widely over urban and rural environments, requiring a method that is cost-effective and scalable so that many measurements can be made across an air shed. We developed such a method that uses passive sampling and light microscopy for analysis. Inexpensive and easy to operate, passive samplers rely on the gravitational settling of particles onto microscope slides. This determines airborne pollen concentration through: 1) sample collection using a modified Durham sampler, 2) preparation of samples for microscopy and strategic sample imaging, and 3) simplified particle measurements and calculation of pollen concentration following deposition velocity models proposed by Scheppegrell [1] and Wagner and Leith [2]. This method was verified with two sampling campaigns during the ragweed season of 2020 and the tree pollen season of 2021. The concentrations determined with the passive and Burkard sampling methods were found to be well-correlated (r > 0.99, r = 0.87) and precise (%CV = 20 %, 21 %). The validation of passive samplers will enable measurements of aeroallergens over wider spatial scales and help determine where aeroallergen exposure risks are greatest.•An inexpensive and low-cost method was developed to determine airborne pollen counts.•The method was evaluated for its accuracy and reproducibility.•The method can be applied to examine the concentrations and spatial variability of airborne pollen.

Preparation of Retinal Samples for Volume Electron Microscopy.

Volume electron microscopy (Volume EM) has emerged as a powerful tool for visualizing the 3D structure of cells and tissues with nanometer-level precision. Within the retina, various types of neurons establish synaptic connections in the inner and outer plexiform layers. While conventional EM techniques have yielded valuable insights into retinal subcellular organelles, their limitation lies in providing 2D image data, which can hinder accurate measurements. For instance, quantifying the size of three distinct synaptic vesicle pools, crucial for synaptic transmission, is challenging in 2D. Volume EM offers a solution by providing large-scale, high-resolution 3D data. It is worth noting that sample preparation is a critical step in Volume EM, significantly impacting image clarity and contrast. In this context, we outline a sample preparation protocol for the 3D reconstruction of photoreceptor axon terminals in the retina. This protocol includes three key steps: retina dissection and fixation, sample embedding processes, and selection of the area of interest.

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.

A deep learning dataset for sample preparation artefacts detection in multispectral high-content microscopy

High-content image-based screening is widely used in Drug Discovery and Systems Biology. However, sample preparation artefacts may significantly deteriorate the quality of image-based screening assays. While detection and circumvention of such artefacts could be addressed using modern-day machine learning and deep learning algorithms, this is widely impeded by the lack of suitable datasets. To address this, here we present a purpose-created open dataset of high-content microscopy sample preparation artefact. It consists of high-content microscopy of laboratory dust titrated on fixed cell culture specimens imaged with fluorescence filters covering the complete spectral range. To ensure this dataset is suitable for supervised machine learning tasks like image classification or segmentation we propose rule-based annotation strategies on categorical and pixel levels. We demonstrate the applicability of our dataset for deep learning by training a convolutional-neural-network-based classifier.

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.

Streptavidin-Affinity Grid Fabrication for Cryo-Electron Microscopy Sample Preparation.

Streptavidin affinity grids provide strategies to overcome many commonly encountered cryo-electron microscopy (cryo-EM) sample preparation challenges, including sample denaturation and preferential orientations that can occur due to the air-water interface. Streptavidin affinity grids, however, are currently utilized by few cryo-EM labs because they are not commercially available and require a careful fabrication process. Two-dimensional streptavidin crystals are grown onto a biotinylated lipid monolayer that is applied directly to standard holey-carbon cryo-EM grids. The high-affinity interaction between streptavidin and biotin allows for the subsequent binding of biotinylated samples that are protected from the air-water interface during cryo-EM sample preparation. Additionally, these grids provide a strategy for concentrating samples available in limited quantities and purifying protein complexes of interest directly on the grids. Here, a step-by-step, optimized protocol is provided for the robust fabrication of streptavidin affinity grids for use in cryo-EM and negative-stain experiments. Additionally, a trouble-shooting guide is included for commonly experienced challenges to make the use of streptavidin affinity grids more accessible to the larger cryo-EM community.

Automated human induced pluripotent stem cell culture and sample preparation for 3D live-cell microscopy.

To produce abundant cell culture samples to generate large, standardized image datasets of human induced pluripotent stem (hiPS) cells, we developed an automated workflow on a Hamilton STAR liquid handler system. This was developed specifically for culturing hiPS cell lines expressing fluorescently tagged proteins, which we have used to study the principles by which cells establish and maintain robust dynamic localization of cellular structures. This protocol includes all details for the maintenance, passage and seeding of cells, as well as Matrigel coating of 6-well plastic plates and 96-well optical-grade, glass plates. We also developed an automated image-based hiPS cell colony segmentation and feature extraction pipeline to streamline the process of predicting cell count and selecting wells with consistent morphology for high-resolution three-dimensional (3D) microscopy. The imaging samples produced with this protocol have been used to study the integrated intracellular organization and cell-to-cell variability of hiPS cells to train and develop deep learning-based label-free predictions from transmitted-light microscopy images and to develop deep learning-based generative models of single-cell organization. This protocol requires some experience with robotic equipment. However, we provide details and source code to facilitate implementation by biologists less experienced with robotics. The protocol is completed in less than 10 h with minimal human interaction. Overall, automation of our cell culture procedures increased our imaging samples' standardization, reproducibility, scalability and consistency. It also reduced the need for stringent culturist training and eliminated culturist-to-culturist variability, both of which were previous pain points of our original manual pipeline workflow.

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.

Copper Oxide Spike Grids for Enhanced Solution Transfer in Cryogenic Electron Microscopy.

The formation of uniform vitreous ice is a crucial step in the preparation of samples for cryogenic electron microscopy (cryo-EM). Despite the rapid technological progress in EM, controlling the thickness of vitreous ice on sample grids with reproducibility remains a major obstacle to obtaining high-quality data in cryo-EM imaging. The commonly employed classical blotting process faces the problem of excess water that cannot be absorbed by the filter paper, resulting in the formation of thick and heterogeneous ice. In this study, we propose a novel approach that combines the recently developed nanowire self-wicking technique with the classical blotting method to effectively control the thickness and homogeneity of vitrified ice. With simple procedures, we generated a copper oxide spike (COS) grid by inducing COSs on commercially available copper grids, which can effectively remove excess water during the blotting procedure without damaging the holey carbon membrane. The ice thickness could be controlled with good reproducibility compared to non-oxidized grids. Incorporated into other EM techniques, our new modification method is an effective option for obtaining high-quality data during cryo-EM imaging.

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.

Fabrication of FeCoNi medium-entropy alloy nanoparticles by high-repetition-rate UV picosecond laser ablation in water

Laser ablation in liquid (LAL) is a rapid heating and cooling process that enables the formation of a variety of nanomaterials. Extensive LAL studies for metals, semiconductors, insulators, and carbons have been reported. Further, alloys are worthwhile target to investigate the applicability of LAL. We report the LAL of equiatomic FeCoNi medium-entropy alloy (MEA) in water using a high-repetition-rate UV picosecond laser with a galvanometric scanner. The extinction spectra of the colloidal solution, morphology, elemental composition, and size distribution of MEA nanoparticles (NPs) were investigated as a function of laser-irradiation time, laser power, and scanning speed. Electron microscopy revealed that chain-like superstructures longer than 10 µm consisting of Ni-rich FeCoNi MEA-core Fe-shell spherical NPs of approximately 50-nm diameter were formed. These superstructures were not originated from the NP agglomeration during sample preparation for microscopy or the successive laser irradiation of colloids, but from the overstriking ablation on the target surface. Generally, the LAL target was irradiated by spatially and temporally separated near-infrared or visible laser pulses to efficiently produce NPs. Overstriking of UV ultrashort laser pulses was shown to be a valuable strategy for modifying the elemental composition of MEA NPs and for forming their superstructures.

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.

Advancement and challenges in sample preparation for atomic force microscopy and infrared microscopy for wood-based materials.

Wood-based materials such as composites or laminates play an important role in today's furniture industry, especially in manufacturing high-quality kitchen and dining room furniture. One important aspect after fabrication is the investigation of these materials to derive quality metrics such as surface stain and scuff resistance. Current sample preparation methods are mostly straightforward and rely on cutting and grinding the materials under test, including sensitive wood substrates. To investigate cross-sections and different layer topologies, characterisation techniques such as atomic force microscopy (AFM) and infrared (IR) microscopy are of potential interest. However, a huge limitation is that current sample preparation methods lead to smeared coatings on the sample cross-sections and high surface roughness. Hence, these methods are not applicable for the sample preparation in measuring AFM and IR microscopy. Therefore, new preparation techniques need to be developed accordingly. This article presents a new approach towards coated wood-based sample preparation including embedding processes to use those samples for AFM and IR microscopy technologies. The proposed method has been evaluated by obtaining AFM, IR and microscopy measurements of more than four different wood-based samples such as (i) raw paper, (ii) impregnated paper, (iii) melamine-coated chipboards and (iv) medium-density fibreboards. The investigation results showed a significant improvement in sample preparation, as well as clear chemical and physical characterisation over whole sample construction, including coating layers, for wood-based materials.