Nanoporous Gold Thin Films as Substrates to Analyze Liquids by Cryo-atom Probe Tomography.

Cryogenic atom probe tomography (cryo-APT) is being developed to enable nanoscale compositional analyses of frozen liquids. Yet, the availability of readily available substrates that allow for the fixation of liquids while providing sufficient strength to their interface is still an issue. Here, we propose the use of 1-2-µm-thick binary alloy film of gold-silver sputtered onto flat silicon, with sufficient adhesion without an additional layer. Through chemical dealloying, we successfully fabricate a nanoporous substrate, with an open-pore structure, which is mounted on a microarray of Si posts by lift-out in the focused-ion beam system, allowing for cryogenic fixation of liquids. We present cryo-APT results obtained after cryogenic sharpening, vacuum cryo-transfer, and analysis of pure water on the top and inside the nanoporous film. We demonstrate that this new substrate has the requisite characteristics for facilitating cryo-APT of frozen liquids, with a relatively lower volume of precious metals. This complete workflow represents an improved approach for frozen liquid analysis, from preparation of the films to the successful fixation of the liquid in the porous network, to cryo-APT.

The effect of specimen preparation on the nanostructure of SLES aqueous solutions observed by cryo-TEM.

Sodium lauryl ether sulphate (SLES) is a detergent widely used in cosmetics and personal-care industries; hence, it is of particular interest to study the self-assembled nanostructure it forms at different conditions. Cryogenic transmission electron microscopy (cryo-TEM) is the most suitable technique for the direct-imaging of such systems. However, since SLES is sensitive to flow and shear, specimen preparation artefacts may misrepresent the native state of the solution. In this paper, we present different cryo-TEM specimen preparation methods, and show how they affect the nanostructure of the system. In fact, for this system, we were able to record the native state of the solution only after sufficient time of on-the-grid relaxation (OGR) after blotting. Here, we also intend to point out the importance of considering the nature of the solution when preparing cryo-TEM specimens.

Scalable substrate development for aqueous sample preparation for atom probe tomography.

Reliable and consistent preparation of atom probe tomography (APT) specimens from aqueous and hydrated biological specimens remains a significant challenge. One particularly difficult process step is the use of a focused ion beam (FIB) instrument for preparing the required needle-shaped specimen, typically involving a 'lift-out' procedure of a small sample of material. Here, two alternative substrate designs are introduced that enable using FIB only for sharpening, along with example APT datasets. The first design is a laser-cut FIB-style half-grid close to those used for transmission electron microscopy (TEM) that can be used in a grid holder compatible with APT pucks. The second design is a larger, standalone self-supporting substrate called a 'crown', with several specimen positions, which self-aligns in APT pucks, prepared by electrical discharge machining (EDM). Both designs are made nanoporous, to provide strength to the liquid-substrate interface, using chemical and vacuum dealloying. Alpha brass, a simple, widely available, lower-cost alternative to previously proposed substrates, was selected for this work. The resulting designs and APT data are presented and suggestions are provided to help drive wider community adoption.

A simple specimen preparation method for histopathological evaluation of vestibular organs.

Vestibular organs consist of the maculae staticae, which are located in both the utricle and saccule, as well as the semicircular ducts and their ampullas. There have been no reports on specimen preparation methods for vestibular organs, including maculae staticae or semicircular ducts. In this study, we investigated highly reproducible methods of preparing vestibular organ specimens for histopathological examinations. We established a method that allows researchers to observe the utricle and saccule, including otoliths, the ampulla of a semicircular duct, and parts of semicircular ducts. This highly reproducible method is useful for histopathological analysis of mice with symptoms of abnormal equilibrium caused by medical toxicity and genetic modification.

Preserving elemental content in adherent mammalian cells for analysis by synchrotron-based x-ray fluorescence microscopy.

Trace metals play important roles in biological function, and x-ray fluorescence microscopy (XFM) provides a way to quantitatively image their distribution within cells. The faithfulness of these measurements is dependent on proper sample preparation. Using mouse embryonic fibroblast NIH/3T3 cells as an example, we compare various approaches to the preparation of adherent mammalian cells for XFM imaging under ambient temperature. Direct side-by-side comparison shows that plunge-freezing-based cryoimmobilization provides more faithful preservation than conventional chemical fixation for most biologically important elements including P, S, Cl, K, Fe, Cu, Zn and possibly Ca in adherent mammalian cells. Although cells rinsed with fresh media had a great deal of extracellular background signal for Cl and Ca, this approach maintained cells at the best possible physiological status before rapid freezing and it does not interfere with XFM analysis of other elements. If chemical fixation has to be chosen, the combination of 3% paraformaldehyde and 1.5 % glutaraldehyde preserves S, Fe, Cu and Zn better than either fixative alone. When chemically fixed cells were subjected to a variety of dehydration processes, air drying was proved to be more suitable than other drying methods such as graded ethanol dehydration and freeze drying. This first detailed comparison for x-ray fluorescence microscopy shows how detailed quantitative conclusions can be affected by the choice of cell preparation method.

Focused Ion Beam Preparation of Specimens for Micro-Electro-Mechanical System-based Transmission Electron Microscopy Heating Experiments.

Micro-electro-mechanical systems (MEMS)-based heating holders offer exceptional control of temperature and heating/cooling rates for transmission electron microscopy experiments. The use of such devices is relatively straightforward for nano-particulate samples, but the preparation of specimens from bulk samples by focused ion beam (FIB) milling presents significant challenges. These include: poor mechanical integrity and site selectivity of the specimen, ion beam damage to the specimen and/or MEMS device during thinning, and difficulties in transferring the specimen onto the MEMS device. Here, we describe a novel FIB protocol for the preparation and transfer of specimens from bulk samples, which involves a specimen geometry that provides mechanical support to the electron-transparent region, while maximizing the area of that region and the contact area with the heater plate on the MEMS chip. The method utilizes an inclined stage block that minimizes exposure of the chip to the ion beam during milling. This block also allows for accurate and gentle placement of the FIB-cut specimen onto the chip by using simultaneous electron and ion beam imaging during transfer. Preliminary data from Si and Ag on Si samples are presented to demonstrate the quality of the specimens that can be obtained and their stability during in situ heating experiments.

Modern Focused-Ion-Beam-Based Site-Specific Specimen Preparation for Atom Probe Tomography.

Approximately 30 years after the first use of focused ion beam (FIB) instruments to prepare atom probe tomography specimens, this technique has grown to be used by hundreds of researchers around the world. This past decade has seen tremendous advances in atom probe applications, enabled by the continued development of FIB-based specimen preparation methodologies. In this work, we provide a short review of the origin of the FIB method and the standard methods used today for lift-out and sharpening, using the annular milling method as applied to atom probe tomography specimens. Key steps for enabling correlative analysis with transmission electron-beam backscatter diffraction, transmission electron microscopy, and atom probe tomography are presented, and strategies for preparing specimens for modern microelectronic device structures are reviewed and discussed in detail. Examples are used for discussion of the steps for each of these methods. We conclude with examples of the challenges presented by complex topologies such as nanowires, nanoparticles, and organic materials.

Application of polystyrene disk substrates in cellular cultivation methods: generalized specimen preparation protocol for scanning electron microscopy.

Sample preparation for scanning electron microscopy (SEM) may vary by cellular type, composition and method of cultivation. It has been proposed here that a generalized method of sample preparation may be applied for the visualization of bacteria, fungi, and human cellular tissue without modification of protocol between cell types. The following protocol was developed to incorporate polystyrene disk substrates in the simplification of sample preparation for SEM and reduce the possibility of processing artefacts. The proposed method of preparation may be applied to samples grown in either liquid or solid cultural medium regardless of cell type. With the proposed protocol, centrifugation, isolation and critical point drying are not required, therefore increasing specimen integrity. The incorporation of polystyrene disks showed positive cellular adhesion and applications in SEM for bacterial, fungal and human neuronal tissue. In addition, the simplicity of the proposed protocol is highly adaptable and may be further incorporated to visually analyse the effects of antifungals, antibiotics and disease pathogenesis through pathogen-host interactions. The proposed method of specimen preparation was incorporated in either liquid or solid state growth mediums during the cultivation of the selected cellular samples and revealed great promise in the preservation and visualization under the scanning electron microscope.

Revealing local variations in nanoparticle size distributions in supported catalysts: a generic TEM specimen preparation method.

The specimen preparation method is crucial for how much information can be gained from transmission electron microscopy (TEM) studies of supported nanoparticle catalysts. The aim of this work is to develop a method that allows for observation of size and location of nanoparticles deposited on a porous oxide support material. A bimetallic Pt-Pd/Al(2)O(3) catalyst in powder form was embedded in acrylic resin and lift-out specimens were extracted using combined focused ion beam/scanning electron microscopy (FIB/SEM). These specimens allow for a cross-section view across individual oxide support particles, including the unaltered near surface region of these particles. A site-dependent size distribution of Pt-Pd nanoparticles was revealed along the radial direction of the support particles by scanning transmission electron microscopy (STEM) imaging. The developed specimen preparation method enables obtaining information about the spatial distribution of nanoparticles in complex support structures which commonly is a challenge in heterogeneous catalysis.

Cryogenic-temperature electron microscopy direct imaging of carbon nanotubes and graphene solutions in superacids.

Cryogenic electron microscopy (cryo-EM) is a powerful tool for imaging liquid and semiliquid systems. While cryogenic transmission electron microscopy (cryo-TEM) is a standard technique in many fields, cryogenic scanning electron microscopy (cryo-SEM) is still not that widely used and is far less developed. The vast majority of systems under investigation by cryo-EM involve either water or organic components. In this paper, we introduce the use of novel cryo-TEM and cryo-SEM specimen preparation and imaging methodologies, suitable for highly acidic and very reactive systems. Both preserve the native nanostructure in the system, while not harming the expensive equipment or the user. We present examples of direct imaging of single-walled, multiwalled carbon nanotubes and graphene, dissolved in chlorosulfonic acid and oleum. Moreover, we demonstrate the ability of these new cryo-TEM and cryo-SEM methodologies to follow phase transitions in carbon nanotube (CNT)/superacid systems, starting from dilute solutions up to the concentrated nematic liquid-crystalline CNT phases, used as the 'dope' for all-carbon-fibre spinning. Originally developed for direct imaging of CNTs and graphene dissolution and self-assembly in superacids, these methodologies can be implemented for a variety of highly acidic systems, paving a way for a new field of nonaqueous cryogenic electron microscopy.

Theory and New Applications of Ex Situ Lift Out.

The ex situ lift out (EXLO) adhesion forces are reviewed and new applications of EXLO for focused ion beam (FIB)-prepared specimens are described. EXLO is used to manipulate electron transparent specimens on microelectromechanical systems carrier devices designed for in situ electron microscope analysis. A new patented grid design without a support film is described for EXLO. This new slotted grid design provides a surface for holding the specimen in place and also allows for post lift out processing. Specimens may be easily manipulated into a backside orientation to reduce FIB curtaining artifacts with this slotted grid. Large EXLO specimens can be manipulated from Xe+ plasma FIB prepared specimens. Finally, applications of EXLO and manipulation of FIB specimens using a vacuum probe lift out method are shown. The vacuum probe provides more control for placing specimens on the new slotted grids and also allows for easy manipulation into a backside configuration.

Evaluating the Use of Synthetic Replicas for SEM Identification of Bloodstains (with Emphasis on Archaeological and Ethnographic Artifacts).

Some archaeological or ethnographic specimens are unavailable for direct examination using a scanning electron microscope (SEM) due to methodological obstacles or legal issues. In order to assess the feasibility of using SEM synthetic replicas for the identification of bloodstains (BSs) via morphology of red blood cells (RBCs), three fragments of different natural raw material (inorganic, stone; plant, wood; animal, shell) were smeared with peripheral human blood. Afterwards, molds and casts of the bloodstained areas were made using vinyl polysiloxane (VPS) silicone impression and polyurethane (PU) resin casting material, respectively. Then, the original samples and the resulting casts were coated with gold and examined in secondary-electron mode using a high-vacuum SEM. Results suggest that PU resin casts obtained from VPS silicone molds can preserve RBC morphology in BSs, and consequently that synthetic replicas are feasible for SEM identification of BSs on cultural heritage specimens made of natural raw materials. Although the focus of this study was on BSs, the method reported in this paper may be applicable to organic residues other than blood, as well as to the surface of other specimens when, for any reason, the original is unavailable for an SEM.

Fabrication of carbon films with ∼ 500nm holes for cryo-EM with a direct detector device.

Single particle electron cryomicroscopy (cryo-EM) is often performed using EM grids coated with a perforated or holey layer of amorphous carbon. Regular arrays of holes enable efficient cryo-EM data collection and several methods for the production of micropatterned holey-carbon film coated grids have been described. However, a new generation of direct detector device (DDD) electron microscope cameras can benefit from hole diameters that are smaller than currently available. Here we extend a previously proposed method involving soft lithography with a poly(dimethylsiloxane) (PDMS) stamp for the production of holey-carbon film coated EM grids. By incorporating electron-beam (e-beam) lithography and modifying the procedure, we are able to produce low-cost high-quality holey-carbon film coated EM grids with ∼500nm holes spaced 4µm apart centre-to-centre. We demonstrate that these grids can be used for cryo-EM. Furthermore, we show that by applying image shifts to obtain movies of the carbon regions beside the holes after imaging the holes, the contrast transfer function (CTF) parameters needed for calculation of high-resolution cryo-EM maps with a DDD can be obtained efficiently.

Use of permanent marker to deposit a protection layer against FIB damage in TEM specimen preparation.

Permanent marker deposition (PMD), which creates permanent writing on an object with a permanent marker, was investigated as a method to deposit a protection layer against focused ion beam damage. PMD is a simple, fast and cheap process. Further, PMD is excellent in filling in narrow and deep trenches, enabling damage-free observation of high aspect ratio structures with atomic resolution in transmission electron microscopy (TEM). The microstructure, composition, gap filling ability and planarization of the PMD layer were studied using dual beam focused ion beam, transmission electron microscopy, energy dispersive X-ray spectroscopy and electron energy loss spectroscopy. It was found that a PMD layer is basically an amorphous carbon structure, and that such a layer should be at least 65 nm thick to protect a surface against 30 keV focused ion beam damage. We suggest that such a PMD layer can be an excellent protection layer to maintain a pristine sample structure against focused ion beam damage during transmission electron microscopy specimen preparation.

Formalin-fixed stool improves the performance of the Kato-Katz method.

Background and Aim: Parasitic infections are a public health problem worldwide, including in Thailand. An epidemiological survey for helminthiasis based on stool examination uses the Kato-Katz method as recommended by the World Health Organization. Limitations of this method include the need for fresh stool, time requirement, and lack of quality control. The aim of this study was to enhance the efficiency of the Kato-Katz technique using formalin and glycerol solutions and to implement specimen preparation in fieldwork. Materials and Methods: For the Kato-Katz method, stool samples were divided into formalin-fixed and unfixed groups at various time points and processes. Fresh echinostome eggs were added to each stool group. Incubation with glycerol increased the clearing process. Each group was observed and photographed using a light microscope. Parasite eggs were imaged and compared using the standard Kato-Katz method. Results: Visualization of echinostome eggs from formalin-fixed stool slides was significantly better than that from unfixed stool slides (p < 0.01). Stool samples fixed for 7 days retained normal echinostome eggs morphology. Incubation with glycerol for 1 h resulted in increased Kato-Katz performance by digesting the stool content and enhancing egg observation. Moreover, the results of the Kato-Katz method using fixed and fixed stool plus glycerol for natural helminth infection showed good quality of Opisthorchis viverrini and Taenia egg visualization and normal morphology with a clear background of slides. Conclusion: Formalin-fixed stool could be more suitable than fresh stool for the Kato-Katz method.

Cryo-EM sample preparation for high-resolution structure studies.

High-resolution structures of biomolecules can be obtained using single-particle cryo-electron microscopy (SPA cryo-EM), and the rapidly growing number of structures solved by this method is encouraging more researchers to utilize this technique. As with other structural biology methods, sample preparation for an SPA cryo-EM data collection requires some expertise and an understanding of the strengths and limitations of the technique in order to make sensible decisions in the sample-preparation process. In this article, common strategies and pitfalls are described and practical advice is given to increase the chances of success when starting an SPA cryo-EM project.

Scanning Electron Microscopy.

Scanning electron microscopy (SEM) remains distinct in its ability to allow topographical visualization of structures. Key elements to consider for successful examination of biological specimens include appropriate preparative and imaging techniques. Chemical processing induces structural artifacts during specimen preparation, and several factors need to be considered when selecting fixation protocols to reduce these effects while retaining structures of interest. Particular care for proper dehydration of specimens is essential to minimize shrinkage and is necessary for placement under the high-vacuum environment required for routine operation of standard SEMs. Choice of substrate for mounting and coating specimens can reduce artifacts known as charging, and a basic understanding of microscope settings can optimize parameters to achieve desired results. This article describes fundamental techniques and tips for routine specimen preparation for a variety of biological specimens, preservation of labile or fragile structures, immune-labeling strategies, and microscope imaging parameters for optimal examination by SEM. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Chemical preparative techniques for preservation of biological specimens for examination by SEM Alternate Protocol 1: Practical considerations for the preparation of soft tissues Alternate Protocol 2: Removal of debris from the exoskeleton of invertebrates Alternate Protocol 3: Fixation of colonies grown on agar plates Alternate Protocol 4: Stabilization of polysaccharide structures with alcian blue and lysine Alternate Protocol 5: Preparation of non-adherent particulates in solution for SEM Support Protocol 1: Application of thin layer of adhesive on substrate to improve adherence Support Protocol 2: Poly-L-lysine coating specimen substrates for improved adherence Support Protocol 3: Microwave processing of biological specimens for examination by SEM Basic Protocol 2: Critical point drying of specimens Alternate Protocol 6: Chemical alternative to critical point drying Basic Protocol 3: Sputter coating Alternate Protocol 7: Improved bulk conductivity through "OTOTO" Basic Protocol 4: Immune-labeling strategies Alternate Protocol 8: Immune-labeling internal antigens with small gold probes Alternate protocol 9: Quantum dot or fluoronanogold preparations for correlative techniques Basic Protocol 5: Exposure of internal structures by mechanical fracturing Basic Protocol 6: Exposure of internal structures of tissues by fracturing with liquid nitrogen Basic Protocol 7: Anaglyph production from stereo pairs to produce 3D images.

An apparatus for plasma cleaning and storage of transmission electron microscopy specimens and specimen holders.

The transmission electron microscopy (TEM) specimen with thickness in nanometer scale is susceptible to hydrocarbon contamination and oxidation, and the specimen holder is also susceptible to contaminants, which would deteriorate the quality of TEM imaging and degrade the efficiency of TEM experiments. Conventional pretreatment devices often have limited functions and low practicability, which may cause problems for TEM specimens and holders. In this work, a multifunctional apparatus for plasma cleaning and storage of TEM specimens and specimen holders is developed based on the specific design of the vacuum joints. The apparatus includes a plasma cleaning system, holder storage station, and specimen storage station, which share the same vacuum system. The cleaning of hydrocarbon contaminants on the specimen and storage of the specimens and holders can be achieved simultaneously in this apparatus. TEM imaging and energy-dispersive X-ray spectroscopy (EDS) analyses of two treated specimens using the apparatus demonstrated that it could effectively remove hydrocarbon contaminants on the specimen. The holder storage station, used to preserve TEM holders in vacuum conditions, can also be modified as a specimen storage station by an appropriate design of the specimen storage platform, in which specimens are protected from water and contaminations. The designed apparatus not only robustly avoids damage to the ultrathin specimen and holders but also improves the working efficiency and reduces costs. These advantages could make our apparatus more appealing for the complement to the present commercial plasma cleaning and storage devices. HIGHLIGHTS: An apparatus for the pretreatment of transmission electron microscopy (TEM) specimens and specimen holders with three functions-plasma cleaning, holder storage, and specimen storage-was designed and fabricated. Using this single apparatus, the cleaning of hydrocarbon contaminants on the specimen and storage of the specimens and holders can be achieved simultaneously. The designed apparatus can not only robustly avoid damage to the ultrathin specimen and holders but also improve the working efficiency and reduce costs by adopting a single vacuum system. These advantages could make our apparatus more appealing for the complement to the present commercial plasma cleaning and storage devices.

Addressing compressive deformation of proteins embedded in crystalline ice.

For cryoelectron microscopy (cryo-EM), high cooling rates have been required for preparation of protein samples to vitrify the surrounding water and avoid formation of damaging crystalline ice. Whether and how crystalline ice affects single-particle cryo-EM is still unclear. Here, single-particle cryo-EM was used to analyze three-dimensional structures of various proteins and viruses embedded in crystalline ice formed at various cooling rates. Low cooling rates led to shrinkage deformation and density distortions on samples having loose structures. Higher cooling rates reduced deformations. Deformation-free proteins in crystalline ice were obtained by modifying the freezing conditions, and reconstructions from these samples revealed a marked improvement over vitreous ice. This procedure also increased the efficiency of cryo-EM structure determinations and was essential for high-resolution reconstructions.

Nanofluidic chips for cryo-EM structure determination from picoliter sample volumes.

Cryogenic electron microscopy has become an essential tool for structure determination of biological macromolecules. In practice, the difficulty to reliably prepare samples with uniform ice thickness still represents a barrier for routine high-resolution imaging and limits the current throughput of the technique. We show that a nanofluidic sample support with well-defined geometry can be used to prepare cryo-EM specimens with reproducible ice thickness from picoliter sample volumes. The sample solution is contained in electron-transparent nanochannels that provide uniform thickness gradients without further optimisation and eliminate the potentially destructive air-water interface. We demonstrate the possibility to perform high-resolution structure determination with three standard protein specimens. Nanofabricated sample supports bear potential to automate the cryo-EM workflow, and to explore new frontiers for cryo-EM applications such as time-resolved imaging and high-throughput screening.