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.

Array tomography of in vivo labeled synaptic receptors.

Array tomography (AT) allows one to localize sub-cellular components within the structural context of cells in 3D through the imaging of serial sections. Using this technique, the z-resolution can be improved physically by cutting ultra-thin sections. Nevertheless, conventional immunofluorescence staining of those sections is time consuming and requires relatively large amounts of costly antibody solutions. Moreover, epitopes are only readily accessible at the section's surface, leaving the volume of the serial sections unlabeled. Localization of receptors at neuronal synapses in 3D in their native cellular ultrastructural context is important for understanding signaling processes. Here, we present in vivo labeling of receptors via fluorophore-coupled tags in combination with super-resolution AT. We present two workflows where we label receptors at the plasma membrane: first, in vivo labeling via microinjection with a setup consisting of readily available components and self-manufactured microscope table equipment and second, live receptor labeling by using a cell-permeable tag. To take advantage of a near-to-native preservation of tissues for subsequent scanning electron microscopy (SEM), we also apply high-pressure freezing and freeze substitution. The advantages and disadvantages of our workflows are discussed.

Electron Microscopy of Cryptococcus neoformans: Processing Challenges to Avoid Artifacts.

This chapter describes methodological details for preparing specimens of Cryptococcus neoformans (although it can be applied to any species of the genus) and their subsequent analysis by scanning and transmission electron microscopy. Adaptations to conventional protocols for better preservation of the sample, as well as to avoid artifacts, are presented. The protocols may be used to examine both the surface ultrastructure and the interior of this pathogenic fungus in detail.

Scanning Electron Microscopy of Ctenophores: Illustrative Atlas.

Scanning electron microscopy (SEM) is a powerful tool for ultrastructural analyses of biological specimens at their surface. With comb jellies being very soft and full of water, many methodological difficulties limit their microanatomical studies via SEM. Here, we describe SEM protocols and approaches successfully tested on ctenophores Pleurobrachia bachei and Beroe abyssicola. Our SEM investigation revealed the astonishing diversity of ciliated structures in all major functional systems, different receptor types, and complex muscular architecture. These protocols can also be practical for various basal bilaterian lineages such as cnidarians.

Gelling properties of acid-induced tofu (soybean curd): Effects of acid type and nano-fish bone.

The effects of different types of acid coagulants and nano fish bone (NFB) additives on the characteristics of tofu were investigated using texture analyzers, SEM, FT-IR, and other techniques. The breaking force and penetration distance, in descending order, were found in the tofu induced by glucono-d-lactone (GDL) (180.27 g and 0.75 cm), citric acid (152.90 g and 0.74 cm), lactic acid (123.33 g and 0.73 cm), and acetic acid (69.84 g and 0.58 cm), respectively. The syneresis of these tofu samples was in the reverse order (35.00, 35.66, 39.66, and 44.50%). Lightness and whiteness were not significantly different among the different samples. Regardless of the acid type, the soluble calcium content in the soybean milk was significantly increased after adding NFB. As a result, the breaking force and penetration distance of all tofu samples increased significantly, but the syneresis decreased. Compared with tofu coagulated by other acids, GDL tofu formed a more uniform and dense gel network maintained by the highest intermolecular forces (especially hydrophobic interactions). Regarding the secondary structure, the lowest percentage of α-helix (22.72%) and, correspondingly, the highest ß-sheet (48.32%) and random coil (18.81%) were noticed in the GDL tofu. The effects of NFB on the tofu characteristics can be explained by the changes in the gel network, intermolecular forces, and secondary structure, which were in line with the acid type. The characteristics of acid-induced tofu can be most synergistically improved by coagulation with GDL and NFB.

Rapid Freezing and Cryo-SEM-EDS Imaging of Foraminifera (Unicellular Eukaryotes) Using a Conductive Viscous Cryogenic Glue.

Spatial distribution of water-soluble molecules and ions in living organisms is still challenging to assess. Energy-dispersive X-ray spectroscopy (EDS) via cryogenic scanning electron microscopy (cryo-SEM) is one of the promising methods to study them without loss of dissolved contents. High-resolution cryo-SEM-EDS has challenges in sample preparation, including cross-section exposure and sample drift/charging due to insulative surrounding water. The former becomes problematic for large and inseparable organisms, such as benthic foraminifera, a unicellular eukaryote playing significant roles in marine ecosystems, which often exceed the size limit for the most reliable high-pressure freezing. Here we show graphite oxide dispersed in sucrose solution as a good glue to freeze, expose cross-section by cryo-ultramicrotome, and analyze elemental distribution owing to the glue's high viscosity, adhesion force, and electron conductivity. To demonstrate the effectiveness and applicability of the glue for cryo-SEM-EDS, deep-sea foraminifer Uvigerina akitaensis was sampled during a cruise and plunge frozen directly on the research vessel, where the liquid nitrogen supply is limited. The microstructures were preserved as faithfully in cryo-SEM images as those with the conventional resin-substituted transmission electron micrograph. We found elements colocalized within the cytoplasm originating from water-soluble compounds that can be lost with conventional dehydrative fixation.

Indirect Correlative Light and Electron Microscopy (iCLEM): A Novel Pipeline for Multiscale Quantification of Structure From Molecules to Organs.

Correlative light and electron microscopy (CLEM) methods are powerful methods that combine molecular organization (from light microscopy) with ultrastructure (from electron microscopy). However, CLEM methods pose high cost/difficulty barriers to entry and have very low experimental throughput. Therefore, we have developed an indirect correlative light and electron microscopy (iCLEM) pipeline to sidestep the rate-limiting steps of CLEM (i.e., preparing and imaging the same samples on multiple microscopes) and correlate multiscale structural data gleaned from separate samples imaged using different modalities by exploiting biological structures identifiable by both light and electron microscopy as intrinsic fiducials. We demonstrate here an application of iCLEM, where we utilized gap junctions and mechanical junctions between muscle cells in the heart as intrinsic fiducials to correlate ultrastructural measurements from transmission electron microscopy (TEM), and focused ion beam scanning electron microscopy (FIB-SEM) with molecular organization from confocal microscopy and single molecule localization microscopy (SMLM). We further demonstrate how iCLEM can be integrated with computational modeling to discover structure-function relationships. Thus, we present iCLEM as a novel approach that complements existing CLEM methods and provides a generalizable framework that can be applied to any set of imaging modalities, provided suitable intrinsic fiducials can be identified.

Serial Block Face-Scanning Electron Microscopy as a Burgeoning Technology.

Serial block face scanning electron microscopy (SBF-SEM), also referred to as serial block-face electron microscopy, is an advanced ultrastructural imaging technique that enables three-dimensional visualization that provides largerx- and y-axis ranges than other volumetric EM techniques. While SEM is first introduced in the 1930s, SBF-SEM is developed as a novel method to resolve the 3D architecture of neuronal networks across large volumes with nanometer resolution by Denk and Horstmann in 2004. Here, the authors provide an accessible overview of the advantages and challenges associated with SBF-SEM. Beyond this, the applications of SBF-SEM in biochemical domains as well as potential future clinical applications are briefly reviewed. Finally, the alternative forms of artificial intelligence-based segmentation which may contribute to devising a feasible workflow involving SBF-SEM, are also considered.

PVP solid dispersions containing Poloxamer 407 or TPGS for the improvement of ursolic acid release

Abstract Solid dispersions (SDs) of ursolic acid (UA) were developed using polyvinylpyrrolidone K30 (PVP K30) in combination with non-ionic surfactants, such as D-α-tocopherol polyethylene glycol 1000 succinate (TPGS) or poloxamer 407 (P407) with the aim of enhancing solubility and in vitro release of the UA. SDs were investigated using a 24 full factorial design, subsequently the selected formulations were characterized for water solubility, X-ray diffractometry (XRD), differential scanning calorimetry (DSC), particle diameter, scanning electron microscopy, drug content, physical-chemical stability and in vitro release profile. SDs showed higher UA water-solubility than physical mixtures (PMs), which was attributed by transition of the drug from crystalline to amorphous or molecular state in the SDs, as indicated by XRD and DSC analyses. SD1 (with P407) and SD2 (with TPGS) were chosen for further investigation because they had higher drug load. SD1 proved to be more stable than SD2, revealing that P407 contributed to ensure the stability of the UA. Furthermore, SD1 and SD2 increased UA release by diffusion and swelling-controlled transport, following the Weibull model. Thus, solid dispersions obtained with PVP k-30 and P407 proved to be advantageous to enhance aqueous solubility and stability of UA.

Electrochemical immunosensing of low-density lipoprotein based on sol-gel encapsulation

Abstract Lipoprotein monitoring is desirable in the management of medical conditions such as atherosclerotic cardiovascular disease and coronary artery disease, in which controlling the concentration of these chylomicrons is crucial. Current clinical methods are complex and present poor reproducibility between laboratories. For these reasons, recent guidelines discard the assessment of low-density lipoprotein cholesterol (LDL-C) as a routine analysis during lipid-lowering therapies. Concerning the importance of monitoring this parameter, the authors present an electrochemical immunosensor constructed from a simple and easy-to-reproduce platform that allows detecting and quantifying LDL nanoparticles directly from human serum samples. The performance of the biosensor was studied by scanning electron microscopy, cyclic voltammetry, and electrochemical impedance spectroscopy. The biosensing platform displays good stability and linearity between 30 mg dL-1 and 135 mg dL-1 with a detection limit of 20 mg dL-1. The proposed biosensor can be easily employed for monitoring LDL concentration in clinical treatments.

Development of microparticles and microparticulated tablets containing piperine

Abstract Piper nigrum (black pepper) is used in Indian traditional medicine and its main alkaloid, Piperine (PIP), presents antioxidant, antitumor and neuroprotective pharmacological properties. This substance is insoluble in aqueous media and can irritate the gastrointestinal tract. Aiming to avoid these inconvenient characteristics and enable PIP oral administration, this study suggested the PIP microencapsulation through the emulsion-solvent evaporation method and the preparation of microparticulated tablets by direct compression. An UV-spectroscopy method was validated to quantify PIP. Microparticles and microparticulated tablets were successfully obtained and the microparticles exhibited excellent flow. The scanning electron microscopy images showed that PIP microparticles were intact after compression. The in vitro release showed a controlled release of PIP from microparticles and PIP microparticles from tablets in comparison to PIP and PIP tablets. The release profiles of PIP microparticles and the microparticulated tablets were similar. Therefore, tablets containing PIP microparticles are promising multiparticulated dosage forms because a tablet allows microparticles administration and the intact ones promote a controlled release, decreasing its irritating potential on the mucosa.

Development of inclusion complex based on cyclodextrin and oxazolidine derivative

Abstract Oxazolidine derivatives (OxD) have been described as third-line antibiotics and antitumoral agents. The inclusion complexes based on cyclodextrin could improve the solubility and bioavailability of these compounds. A novel synthetic OxD was used, and its inclusion complexes were based on 2-hydroxy-beta-cyclodextrin (2-HPßCD). We conducted an in silico study to evaluate the interaction capacity between OxD and 2-HPßCD. Characterization studies were performed through scanning electron microscopy (SEM), Fourier-transformed infrared (FTIR), nuclear magnetic resonance spectroscopy (1H-NMR), X-ray diffraction (XRD), and thermal analyses. A kinetic study of the OxD was performed, including a cytotoxicity assay using peripheral blood mononuclear cells (PBMCs). The maximum increment of solubility was obtained at 70 mM OxD using 400 mM 2-HPßCD. SEM analyses and FTIR spectra indicated the formation of inclusion complexes. 1H-NMR presented chemical shifts that indicated 1:1 stoichiometry. Different thermal behaviors were obtained. The pharmacokinetic profile showed a short release time. Pure OxD and its inclusion complex did not exhibit cytotoxicity in PBMCs. In silico studies provided a foremost insight into the interactions between OxD and 2-HPßCD, including a higher solubility in water and an average releasing profile without toxicity in normal cells

Examining atherosclerotic lesions in three dimensions at the nanometer scale with cryo-FIB-SEM.

We employed in a correlative manner an unconventional combination of methods, comprising cathodoluminescence, cryo-scanning electron microscopy (SEM), and cryo-focused ion beam (FIB)-SEM, to examine the volumes of thousands of cubed micrometers from rabbit atherosclerotic tissues, maintained in close-to-native conditions, with a resolution of tens of nanometers. Data from three different intralesional regions, at the media-lesion interface, in the core, and toward the lumen, were analyzed following segmentation and volume or surface representation. The media-lesion interface region is rich in cells and lipid droplets, whereas the core region is markedly richer in crystals and has lower cell density. In the three regions, thin crystals appear to be associated with intracellular or extracellular lipid droplets and multilamellar bodies. Large crystals are independently positioned in the tissue, not associated with specific cellular components. This extensive evidence strongly supports the idea that the lipid droplet surfaces and the outer membranes of multilamellar bodies play a role in cholesterol crystal nucleation and growth and that crystal formation occurs, in part, inside cells. The correlative combination of methods that allowed the direct examination of cholesterol crystals and lipid deposits in the atherosclerotic lesions may be similarly used for high-resolution examination of other tissues containing pathological or physiological cholesterol deposits.

Evaluation of Zebrafish Toxicology and Biomedical Potential of Aeromonas hydrophila Mediated Copper Sulfide Nanoparticles.

The present study deals with extracellular synthesis and characterization of copper sulfide (CuS) nanoparticles using Aeromonas hydrophila, and the biological applications of the synthesized CuS like antibacterial, anti-inflammatory, and antioxidant activity were reported. Further, the toxicological effects of the CuS were evaluated using zebrafish as an animal model. The primary step of the synthesis was carried out by adding the precursor copper sulfates to the culture supernatant of Aeromonas hydrophila. The UV-visible spectrophotometer was used to characterize the synthesized nanoparticles, and the peak was obtained at 307 nm through the reduction process. Fourier transform infrared spectroscopy (FTIR) was involved to find out the functional groups (carboxylic acid, alcohols, alkanes, and nitro compounds) associated with copper sulfide nanoparticles (CuS-NPs). Atomic force microscopy (AFM) was used to characterize the CuS topographically, and a scanning electron microscope (SEM) revealed about 200 nm sized CuS nanoparticles with agglomerated structures. Overall, the characterized nanoparticles can be considered as a potential candidate with therapeutic proficiencies as antibacterial, antioxidant, and anti-inflammatory mediator/agents.

Combined molecular and supramolecular structural insights into pasting behaviors of starches isolated from native and germinated waxy brown rice.

In order to provide a theoretical basis on how germination treatment modulated the pasting behaviors of starches from native and germinated waxy brown rice, impacts of germination on the molecular and supramolecular structures and pasting behaviors were evaluated. Multiple analytical methods such as thermodynamics and spectroscopy were applied in this work for investigating the changes in starch multiscale structure and pasting behaviors. The results show that germination treatment contributed to an obvious increase in α- and ß-amylase activities, which could degrade the starch chains and reduce the contents of double helices (50.9%-43.2%), short-range ordered degree (1.054-0.908), relative crystallinity (40.1%-30.5%), and lamellar ordering degree (dc, 6.09-5.46 nm) along with apparent erosion on starch granules. These structural amorphizations at the molecular and supramolecular levels could lead to the weakened entanglements and interactions among molecular chains, eventually reducing the characteristic viscosity (e.g., overall, peak, and final viscosity) of starch. This study may facilitate better development of germinated rice-based products.

The use of Dalmatian pyrethrum daisy and an excipient in the treatment of seborrheic dermatitis.

The active ingredients of the Pyretrin-D trichological cosmetic series, namely benzyl benzoate, Dalmatian pyrethrum daisy, Cistus incanus, tea tree oil and geranium oil, almond acid and arginine were tested in respect to the treatment of seborrheic dermatitis. The paper describes the application of Dalmatian pyrethrum daisy and the excipient. Methods and devices used to confirm the effectiveness of the tested formulations included the TrichoScope Polarizer Dino-Lite (MEDL4HM) and the scanning electron microscope (SEM).

Immunofluorescence and High-Resolution Microscopy Reveal New Insights in Human Globozoospermia.

Globozoospermia is a rare and severe type of teratozoospermia characterized by the presence of round-headed, acrosomeless spermatozoa with cytoskeleton defects. Current data support a negative relationship between globozoospermia and intracytoplasmic sperm injection (ICSI) outcomes, revealing the need to perform exhaustive studies on this type of sperm disorder. The aim of this study was to evaluate different structural, functional and molecular sperm biomarkers in total globozoospermia with proper embryo development after ICSI. The combination of field-emission scanning electron microscopy (FE-SEM) and transmission electron microscopy (TEM) allowed us to identify and correlate eight morphological patterns with both types of microscopy. Additionally, results reported a high percentage of coiled forms, with cytoplasmic retentions around the head and midpiece. By fluorescent microscopy, we detected that most of the sperm showed tubulin in the terminal piece of the flagellum and less than 1% displayed tyrosine phosphorylation in the flagellum. Moreover, we did not detect chaperone Heat shock-related 70 kDa protein 2 (HSPA2) in 85% of the cells. Overall, these findings provide new insights into globozoospermia, which could have potential implications in improving sperm selection methods for assisted reproductive techniques.

The Influence of the Material Structure on the Mechanical Properties of Geopolymer Composites Reinforced with Short Fibers Obtained with Additive Technologies.

Additive manufacturing technologies have a lot of potential advantages for construction application, including increasing geometrical construction flexibility, reducing labor costs, and improving efficiency and safety, and they are in line with the sustainable development policy. However, the full exploitation of additive manufacturing technology for ceramic materials is currently limited. A promising solution in these ranges seems to be geopolymers reinforced by short fibers, but their application requires a better understanding of the behavior of this group of materials. The main objective of the article is to investigate the influence of the microstructure of the material on the mechanical properties of the two types of geopolymer composites (flax and carbon-reinforced) and to compare two methods of production of geopolymer composites (casting and 3D printing). As raw material for the matrix, fly ash from the Skawina coal power plant (located at: Skawina, Lesser Poland, Poland) was used. The provided research includes mechanical properties, microstructure investigations with the use of scanning electron microscope (SEM), confocal microscopy, and atomic force microscope (AFM), chemical and mineralogical (XRD-X-ray diffraction, and XRF-X-ray fluorescence), analysis of bonding in the materials (FT-IR), and nuclear magnetic resonance spectroscopy analysis (NMR). The best mechanical properties were reached for the sample made by simulating 3D printing process for the composite reinforced by flax fibers (48.7 MPa for the compressive strength and 9.4 MPa for flexural strength). The FT-IR, XRF and XRD results show similar composition of all investigated materials. NMR confirms the presence of SiO4 and AlO4 tetrahedrons in a three-dimensional structure that is crucial for geopolymer structure. The microscopy observations show a better coherence of the geopolymer made in additive technology to the reinforcement and equal fiber distribution for all investigated materials. The results show the samples made by the additive technology had comparable, or better, properties with those made by a traditional casting method.

Effect of Twinlight Laser on the Attachment of Human Gingival Fibroblasts to the Root Surface In Vitro.

BACKGROUND This study aimed to compare the effectiveness of subgingival scaling and root planing with the Twinlight laser, Er: YAG laser, and hand instrumentation on the removal of endotoxin and attachment of human gingival fibroblasts (HGFs) to cementum surfaces in vitro. MATERIAL AND METHODS Single-rooted teeth extracted for periodontal disease were collected and divided into 3 groups: group A, root planing with Gracey curet no. 5/6; group B, irradiation with Er: YAG laser; group C, irradiation with Er: YAG laser and Nd: YAG laser. Endotoxins were determined by the limulus amebocyte lysate test. Cell attachment and proliferation of HGFs on root specimens were evaluated by cell counting kit-8 assay. The root surface and cell morphology were observed by scanning electron microscope. RESULTS A flat root surface with scratches was found in group A, Group B had a homogeneous rough morphology without carbonization, and group C had a non-homogeneous rough morphology with ablation. The endotoxin concentration was highest in group A (P<0.05) and lowest in group C (P>0.05). HGFs cultured in group B showed significantly increased adhesion and proliferation compared with groups A and C (P<0.05). HGFs in group B were well attached, covered densely by pseudopodia. HGFs in group A were round with poor extension and short pseudopodia, while the cells in the group C were in narrow, triangular, or polygonal shapes. CONCLUSIONS Twinlight laser-assisted periodontal treatment effectively improved the biocompatibility of root surface and promoted the attachment and proliferation of fibroblasts by removing calculus and reducing the concentration of endotoxins.

Biofabrication of ZnO nanoparticles using Acacia arabica leaf extract and their antibiofilm and antioxidant potential against foodborne pathogens.

Emergence of multidrug resistant pathogens is increasing globally at an alarming rate with a need to discover novel and effective methods to cope infections due to these pathogens. Green nanoparticles have gained attention to be used as efficient therapeutic agents because of their safety and reliability. In the present study, we prepared zinc oxide nanoparticles (ZnO NPs) from aqueous leaf extract of Acacia arabica. The nanoparticles produced were characterized through UV-Visible spectroscopy, scanning electron microscopy, and X-ray diffraction. In vitro antibacterial susceptibility testing against foodborne pathogens was done by agar well diffusion, growth kinetics and broth microdilution assays. Effect of ZnO NPs on biofilm formation (both qualitatively and quantitatively) and exopolysaccharide (EPS) production was also determined. Antioxidant potential of green synthesized nanoparticles was detected by DPPH radical scavenging assay. The cytotoxicity studies of nanoparticles were also performed against HeLa cell lines. The results revealed that diameter of zones of inhibition against foodborne pathogens was found to be 16-30 nm, whereas the values of MIC and MBC ranged between 31.25-62.5 µg/ml. Growth kinetics revealed nanoparticles bactericidal potential after 3 hours incubation at 2 × MIC for E. coli while for S. aureus and S. enterica reached after 2 hours of incubation at 2 × MIC, 4 × MIC, and 8 × MIC. 32.5-71.0% inhibition was observed for biofilm formation. Almost 50.6-65.1% (wet weight) and 44.6-57.8% (dry weight) of EPS production was decreased after treatment with sub-inhibitory concentrations of nanoparticles. Radical scavenging potential of nanoparticles increased in a dose dependent manner and value ranged from 19.25 to 73.15%. Whereas cytotoxicity studies revealed non-toxic nature of nanoparticles at the concentrations tested. The present study suggests that green synthesized ZnO NPs can substitute chemical drugs against antibiotic resistant foodborne pathogens.