High Vacuum Scanning Electron Microscopy Research Articles

Optimized protocol for high-vacuum scanning electron microscopy analysis of polyacrylamide hydrogel-attached sperm cells in a binary system.

A protocol for the analysis of a binary system comprising polyacrylamide hydrogel-attached sperm cells using high-vacuum scanning electron microscopy (SEM) is presented. This protocol focuses on optimizing the SEM procedure to obtain accurate and detailed imaging of the sperm cells and their interactions with the hydrogel scaffold. The methodology involves a stepwise sample preparation, including sample dehydration through a gradual exchange of ethanol/water ratios, followed by the application of a conductive metal coating. By employing this modified protocol, the traditional use of acetone dehydration, which may introduce chemical alterations to the materials, is avoided. The proposed approach enables a comprehensive evaluation of the morphology and interactions within the biological system in contact with the soft material scaffold. Furthermore, the potential application of this protocol extends to the study of other mammalian reproductive cells or cells of different origins adhered to hydrogel scaffolds. RESEARCH HIGHLIGHTS: Novel SEM protocol reveals precise imaging of sperm-hydrogel attachment in a binary system, enhancing our understanding of cell-material interactions. By optimizing SEM procedures, the protocol achieves precise imaging of sperm-hydrogel interactions using ethanol/water dehydration and a conductive metal coating. This modified approach enables a thorough assessment of morphology and interactions in the binary system,extending its potential applicability to other reproductive cells on hydrogelscaffolds.

Smear Layer Removal Using Passive Ultrasonic Irrigation and Different Concentrations of Sodium Hypochlorite

IntroductionThe present study evaluated the effect of passive ultrasonic activation (PUI) of EDTA solution followed by conventional irrigation with 2 concentrations of sodium hypochlorite (NaOCl) on smear layer removal. MethodsFifty single-root mandibular premolars were chemomechanically prepared with ProTaper Universal (Dentsply Maillefer, Ballaigues, Switzerland) nickel-titanium rotary instruments and 1% NaOCl. The roots were cleaved, and the dentin surfaces were analyzed with scanning electron microscopy operated at a low vacuum. Images were obtained from previously demarcated areas in each root canal third. The teeth were reassembled and distributed into 5 groups according to the final irrigation protocols (n = 10): group 1, EDTA/PUI + 1% NaOCl; group 2, EDTA/PUI + 5% NaOCl; group 3, EDTA/CI + 1% NaOCl; group 4, EDTA/CI + 5% NaOCl; and group 5 (negative control), saline solution/PUI. After irrigation, the teeth were reseparated and prepared for conventional high-vacuum scanning electron microscopy of the same dentin surface that was previously analyzed. The amount of debris was classified using a 4-point scoring system. Data were analyzed with Kruskal-Wallis and Dunn tests at α = 0.05. ResultsGroups 1 and 2, which used PUI and different concentrations of NaOCl, were not significantly different; however, they differed significantly from group 3 (P < .05). With respect to canal cleanliness at different root thirds, all groups showed the worst cleaning at the apical third. ConclusionsPUI activation of the EDTA irrigant is required when canal debridement is performed with EDTA and a lower concentration of NaOCl.

Environmental gas impact on the emission volume of X-rays near the interface in the variable pressure scanning electron microscopy

In this paper, our previously developed model to account for the secondary X-ray fluorescence and absorption effects near the interface of two adjacent materials in a high-vacuum scanning electron microscope (Zoukel & Khouchaf, 2014) is adapted and extended to experimental conditions of low-vacuum mode (in the presence of a gaseous environment in the SEM analysis chamber). The position shifting effect of the two Gaussian peaks issued from the first derivative equation that can fit the experimental low-vacuum EDS profiles is investigated. The impact of the medium gas on the emission volume of secondary X-rays near the interface is qualitatively discussed. Water vapour and helium are successively used as gas environment, in order to link the resolution of microanalysis profiles with the effects of the X-ray fluorescence and absorption phenomenon. A close agreement between Monte Carlo simulation and experimental results is found.

Skeleton bones in museum indoor environments offer niches for fungi and are affected by weathering and deposition of secondary minerals.

Large skeleton specimens are often featured as iconic open displays in Natural History Museums, for example, the blue whale 'Hope' at the Natural History Museum, London. A study on Hope's bone surface was performed to assess the biodeterioration potential of fungi. Fungi were isolated, and a fungal internal transcribed spacer (ITS) clone library survey was performed on dust and bone material. Mineral particles derived from bone and dust were analysed using energy dispersive X-ray spectroscopy, variable pressure scanning electron microscopy (SEM) and high vacuum SEM. Results showed that bone material, although mainly mineral in nature, and therefore less susceptible than organic materials to biodeterioration phenomena in the indoor environments, offers niches for specialized fungi and is affected by unusual and yet not so well-documented mechanisms of alteration. Areas of bone surface were covered with a dense biofilm mostly composed of fungal hyphae, which produced tunnelling and extensive deposition of calcium and iron-containing secondary minerals. Airborne halophilic and xerophilic fungi including taxa grouping into Ascomycota and Basidiomycota, capable of displacing salts and overcome little water availability, were found to dominate the microbiome of the bone surface.

In-Situ Characterization of Lithium Native Passivation Layer in A High Vacuum Scanning Electron Microscope.

A technique to characterize the native passivation layer (NPL) on pure lithium metal foils in a scanning electron microscope (SEM) is described in this paper. Lithium is a very reactive metal, and consequently, observing and quantifying its properties in a SEM is often compromised by rapid oxidation. In this work, a pure lithium energy-dispersive x-ray spectrum is obtained for the first time in a high vacuum SEM using a cold stage/cold trap with liquid nitrogen reservoir outside the SEM chamber. A nanomanipulator (OmniProbe 400) inside the microscope combined with x-ray microanalysis and windowless energy dispersive spectrometer is used to fully characterize the NPL of lithium metal and some of its alloys by a mechanical removal procedure. The results show that the native films of pure lithium and its alloys are composed of a thin (25 nm) outer layer that is carbon-rich and an inner layer containing a significant amount of oxygen. Differences in thickness between laminated and extruded samples are observed and vary depending on the alloy composition.

Sulfate–nitrate–ammonium as double salts in PM2.5: Direct observations and implications for haze events

Obtaining detailed information on sulfate–nitrate–ammonium (SNA) is fundamentally important to explain the formation of haze in China, since it is a dominant component of fine particulate matter (PM2.5) and plays a critical role in the deterioration of air quality. Several single-particle analysis methods have been applied to study and explain SNA formation; however, determining its mixture state remains a challenge. This study describes a direct observation of the SNA components in atmospheric particles on a single-particle scale, and details the first use of a non-destructive surface-enhanced Raman scattering (SERS) technique for SNA analysis. We studied PM2.5 collected at a site on the premises of Tsinghua University in Beijing, China, during a winter haze episode (12.15.2016–12.23.2016). The on-line data show that the SNA component accounted for 9.4% to 68.2% of the total mass of PM2.5, becoming dominant on heavy haze days, and the sulfate concentration increased with the nitrate concentration (R2 = 0.72). Furthermore, the off-line SERS and scanning electron microscopy-energy dispersive X ray analysis (SEM-EDS) results for the single particles collected also indicated that SNA increase with increasing haze pollution. The existing state of the SNA component on each haze day was observed directly in a non-destructive manner mainly in the form of double salts such as 3(NH4NO3)·(NH4)2SO4 and 2(NH4NO3)·(NH4)2SO4. A Raman mapping experiment further confirmed that the SNA was internally mixed. Our data also show that SNA can evaporate under high-vacuum scanning electron microscopy conditions, suggesting that SERS is an effective method to directly observe SNA without sample loss and may represent a promising single-particle technique to supplement traditional electron microscopy methods. This work will provide evidence for the SNA formation, particularly during haze events.

The effect of THPS concentration on nPAG microstructure

The microstructures formed within a normoxic polyacrylamide gel (nPAG) dosimeter and their correlation to amount of dose received is investigated through the use of two scanning electron microscope modalities. The effect of gold sputtering onto the samples was examined to determine if it impacted the detectable structures. The evolution of polymer structure with increasing dose is shown to progress from long shard like polymer chains to smooth polymer structures coating the gel surface. It is demonstrated that without gold coating the samples, which is a necessity in high vacuum scanning electron microscopy, that an environmental scanning electron microscope can be used for analysis of polymerized structures. The addition of THPS as an antioxidant is shown to not alter the formation of polymer microstructures when compared to other studies involving THPC. It is also proposed that knowledge of these features could be used to manipulate the deformability, stability and polymerized opacity of an nPAG dosimeter.

Hydrogel Micro-/Nanosphere Coated by a Lipid Bilayer: Preparation and Microscopic Probing.

The result of polymeric nanogels and lipid vesicles interaction—lipobeads—can be considered as multipurpose containers for future therapeutic applications, such as targeted anticancer chemotherapy with superior tumor response and minimum side effects. In this work, micrometer sized lipobeads were synthesized by two methods: (i) mixing separately prepared microgels made of poly(N-isopropylacrylamide) (PNIPA) and phospholipid vesicles of micrometer or nanometer size and (ii) polymerization within the lipid vesicles. For the first time, a high vacuum scanning electron microscopy was shown to be suitable for a quick validation of the structural organization of wet lipobeads and their constituents without special sample preparation. In particular, the structural difference of microgels prepared by thermal and UV-polymerization in different solvents was revealed and three types of giant liposomes were recognized under high vacuum in conjunction with their size, composition, and method of preparation. Importantly, the substructure of the hydrogel core and multi- and unilamellar constructions of the peripheral lipid part were explicitly distinguished on the SEM images of lipobeads, justifying the spontaneous formation of a lipid bilayer on the surface of microgels and evidencing an energetically favorable structural organization of the hydrogel/lipid bilayer assembly. This key property can facilitate lipobeads’ preparation and decrease technological expenses on their scaled production. The comparison of the SEM imaging with the scanning confocal and atomic force microscopies data are also presented in the discussion.

Ultrastructural Analysis of the Foreskin in Patients With True Phimosis Treated or Not Treated With Topical Betamethasone and Hyaluronidase Ointment

To evaluate, by using scanning electron microscopy (SEM), the possible alterations of the foreskin connective tissue in patients with true phimosis submitted to topical treatment with betamethasone and hyaluronidase ointment. We studied 15 patients (mean 5.3 years old) submitted or not to topical application of betamethasone 0.2% and hyaluronidase cream. For qualitative analysis of the connective tissue, we studied 5 samples from each foreskin, with 2 mm length. The samples were submitted to fixation for SEM by immersing tissue fragments in a modified Karnovsky solution for 48 hours at 4°C. The obtained acellular preparations were then processed for high-vacuum SEM, and observations were performed on a LEO 435 (Zeiss, Oberkochen, Germany) scanning electron microscope with an acceleration voltage of 15 to 20 kV. Parents of 7 children did not agree with the clinical treatment and opted for circumcision directly. These patients served as the control group (nontreated). Eight patients submitted to topical treatment could not expose the glans and were referred for circumcision. In SEM, with a magnification of 5000×, we observed important differences in the organization of the collagen and elastic system fibers when comparing treated and nontreated patients with betamethasone and hyaluronidase. Treated patients presented a different organization of collagen with a clear decrease in the elastic system fibers. The treatment of phimosis with betamethasone + hyaluronidase showed changes in the structure of the foreskin with a decrease of elastic system fibers, which is characteristic of the healing processes.

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.

Resolving topography of an electron beam-sensitive oxalate-phosphate-amine metal–organic framework (OPA-MOF)

Scanning electron microscopy (SEM) has, for many years, been the favoured method to gain insights into morphology, micro- and surface structure of new materials, thus development of SEM instruments and modes of use has been rapid. Yet, high-quality, charge artefact-free SEM-representation of highly beam-sensitive non-conductive hybrid metal–organic frameworks (MOF) remains challenging, particularly if access to highly specialised instrumentation is limited—a situation many researchers face. This study details a systematic approach taken to determine the appropriate instrument operating conditions and sample preparation methods for characterisation of a oxalate-phosphate-amine MOF (OPA-MOF) under conventional high-vacuum SEM conditions. We show that a double-coating method adapted from biological sciences, where a carbon coating (≤15 nm) is followed by a thin gold coating (~3–5 nm), enables charge- and damage-free imaging of the electron beam-sensitive OPA-MOF. Details of micro-topography are sufficiently resolved for intended purposes (~100 nm) and are not unduly masked by the coating.

Surface and interfacial analysis of sandblasted and acid-etched enamel for bonding orthodontic adhesives.

The purposes of this study were to characterize enamel surfaces treated with sandblasting, acid etching, or sandblasting followed by acid etching for bonding of orthodontic adhesives and to evaluate the enamel-adhesive interfaces. Buccal premolar surfaces were sandblasted, acid etched, sandblasted and subsequently acid etched, or left intact. For 2 treatments (acid etching and sandblasting plus acid etching), orthodontic brackets were bonded for resin infiltration assessment. Surface roughness and enamel loss were evaluated by optical profilometry, and surface morphology and elemental composition were examined by low-vacuum scanning electron microscope/energy dispersive x-ray analysis, whereas interfacial resin infiltration was assessed by high-vacuum scanning electron microscopy. Differences were statistically analyzed by 1-way analysis of variance and Tukey-Kramer multiple comparison tests or t tests (α = 0.05). The sandblasted and sandblasted-plus-acid-etched groups showed higher surface roughness values than the acid-etched group. Sandblasting plus acid etching removed more enamel than did sandblasting or acid etching alone. Sandblasting plus acid etching demonstrated a more uniform type I etching pattern, without alumina particles implanted in the enamel, unlike sandblasting. No difference was found in the extent of resin infiltration between the bonded acid-etched and sandblasted-plus-acid-etched groups, although the latter showed improved infiltration characteristics. Sandblasting-plus-acid-etching treatment creates a rough enamel surface, typical of type I etching, with the greatest extent of enamel loss, free of alumina interferences, demonstrating the same extent of resin infiltration as acid etching, but exhibiting improved infiltration quality.

ナノスーツ法による生きたままの生物のSEM 内生態観察

ナノスーツ法による生きたままの生物のSEM 内生態観察

Measurement of the SEM-beam diameter using a relief structure: Influence of contamination

Relief structures on plate surfaces are used to measure the diameter of the scanning electron microscope (SEM) beam. A contaminant film which alters the geometric parameters of the relief structure appears on its surface even in a high-vacuum SEM. It turns out that the film is deposited onto the surface nonuniformly, and there exist such regions of the film that remain almost unchanged after structure scanning for many hours. The discovered phenomenon is explained in this paper. The values of the beam diameter determined using these regions can be used to monitor the stability of a measuring SEM under conditions of relief-structure surface contamination. The beam diameter is determined using two models. In a model with an a priori unchanged beam form, the variation in the effective diameter value is ∼2 nm during long-term scanning. In a model with the recovery of the beam form, the diameter variation is larger; however, the character of the time dependence is similar to that observed for the effective value in the model with the unchanged beam form.

Interactions of dentine desensitisers with human dentine: Morphology and composition

ObjectiveTo evaluate the effect of desensitising agents on human dentine morphology and composition. MethodsRandomly assigned human coronal-dentine specimens were subjected to: (a) no treatment (smear-layer control, n=4); (b) acid etching with 6% citric acid (demineralised control, n=4); (c) treatment with desensitising agents (12 cycles of 60 s treatment with 60 s between-treatment rinsing, n=6 per agent); and (d) exposure to acidic challenge (pH 5.0 for 90 s, n=6 per agent). The tested products were: Listerine® Advanced Defence Sensitive (LADS; 1.4% potassium oxalate) mouthrinse, Colgate® Sensitive Pro-Relief™ mouthrinse, and toothpaste slurries (paste/water 1:2 wt/wt ratio) of Colgate® Sensitive Pro-Relief™ paste, Crest® Sensitive paste and Sensodyne® Repair and Protect paste. All dentine surfaces were studied by attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, Raman microscopy and high vacuum scanning electron microscopy with energy-dispersive X-ray microanalysis (HV-SEM/EDX). ResultsDesensitising slurry treatments occluded tubule orifices of acid-etched dentine, creating a randomly distributed surface pattern of particle aggregates. The greatest intratubular penetration of occluding particles was found in dentine treated with LADS. The atomic ratios of Ca/N and Ca/P, and the mineral/matrix ratios increased after toothpaste-slurry treatments compared with the acid-etched dentine. However, the acidic challenge removed most surface precipitates and further demineralised these substrates. Before the acidic challenge, the surface features were least affected in specimens treated with Sensodyne® Repair and Protect. After the acidic challenge, the sub-surface occlusion features were least affected in specimens treated with LADS. Clinical significanceAlthough most tested products achieved occlusion of dentinal tubules and provided evidence of mineral deposits, the deposit formed by LADS demonstrated the greatest resistance to acidic challenge, which simulates intra-oral demineralisation phases.

Human Bloodstains on Biological Materials: High-Vacuum Scanning Electron Microscope Examination Using Specimens without Previous Preparation

Studies of human bloodstains on nonbiological materials have been previously carried out using a high-vacuum scanning electron microscope (HV-SEM) in secondary-electron mode without any sample treatment. To assess whether biological substrates can affect the morphology of human erythrocytes in bloodstains, three fragments of different biological material (bone, shell, and wood) were smeared with peripheral human blood. Afterward, the bloodstains were directly examined in secondary-electron mode by an HV-SEM following a procedure initially standardized to be used in uncoated human bloodstains on stone. The obtained results suggest that HV-SEM is suitable for examining untreated bloodstains on biological substrate and that the morphology of erythrocytes in human bloodstains is not affected by the biological nature of the substrate. A cautionary issue regarding bloodstains on nondehydrated biological substrates is that the waiting time required for initiating the HV-SEM examination is by far higher than when using inorganic bloodstain substrates.

Comparison of biofilm and attachment mechanisms of a phytopathological and clinical isolate of Klebsiella pneumoniae Subsp. pneumoniae.

Some bacterial species can colonize humans and plants. It is almost impossible to prevent the contact of clinically pathogenic bacteria with food crops, and if they can persist there, they can reenter the human food chain and cause disease. On the leaf surface, microorganisms are exposed to a number of stress factors. It is unclear how they survive in such different environments. By increasing adhesion to diverse substrates, minimizing environmental differences, and providing protection against defence mechanisms, biofilms could provide part of the answer. Klebsiella pneumoniae subsp. pneumoniae is clinically important and also associated with fruit diseases, such as “pineapple fruit collapse.” We aimed to characterize biofilm formation and adhesion mechanisms of this species isolated from pineapple in comparison with a clinical isolate. No differences were found between the two isolates quantitatively or qualitatively. Both tested positive for capsule formation and were hydrophobic, but neither produced adherence fibres, which might account for their relatively weak adhesion compared to the positive control Staphylococcus epidermidis ATCC 35984. Both produced biofilms on glass and polystyrene, more consistently at 40°C than 35°C, confirmed by atomic force and high-vacuum scanning electron microscopy. Biofilm formation was maintained in an acidic environment, which may be relevant phytopathologically.

Severe Head Lice Infestation in an Andean Mummy of Arica, Chile

Pediculus humanus capitis is an ancient human parasite, probably inherited from pre-hominid times. Infestation appears as a recurrent health problem throughout history, including in pre-Columbian populations. Here, we describe and discuss the occurrence of pre-Columbian pediculosis in the Andean region of the Atacama Desert. Using a light microscope and scanning electron microscopy, we studied a highly infested Maitas Chiribaya mummy from Arica in northern Chile dating to 670-990 calibrated years A.D. The scalp and hair of the mummy were almost completely covered by nits and adult head lice. Low- and high-vacuum scanning electron microscopy revealed a well-preserved morphology of the eggs. In addition, the excellent preservation of the nearly 1,000-yr-old adult head lice allowed us to observe and characterize the head, antennae, thorax, abdomen, and legs. Leg segmentation, abdominal spiracles, and sexual dimorphism also were clearly observed. The preservation of the ectoparasites allowed us to examine the micromorphology using scanning electron microscopy; the opercula, aeropyles, and spiracles were clearly visible. This case study provides strong evidence that head lice were a common nuisance for Andean farmers and herders. Head lice are transmitted by direct head-to-head contact; thus, this ancient farmer and herder was potentially infesting other people. The present study contributes to the body of research focusing on lice in ancient populations.

Vessel elements present in the secondary xylem of Trochodendron and Tetracentron (Trochodendraceae)

For almost 150 years, the two monotypic genera Trochodendron and Tetracentron (Trochodendraceae) have been considered to share an unusual and primitive feature in angiosperms – the lack of vessels in their wood. Therefore, they have been classified in a basal position in the angiosperms. Our observations by light microscopy, low-vacuum environmental scanning electron microscopy (ESEM) and high-vacuum scanning electron microscopy (SEM) both in fresh and FAA-fixed materials consistently showed the presence of tracheary elements differentiated into two types in both genera. In Trochodendron, the tracheary elements can be divided into perforate vessel elements and imperforate fiber-tracheids and tracheids. The vessel elements show end and lateral walls. The pits on the end walls are elongate- broadened and do not have membranes or only a few remnants of them forming the perforation plates. The fiber-tracheids show crossfield pit pairs and sharp ends, and the tracheids show bordered pits. In Tetracentron, the tracheary elements comprise vessel elements and fibers. The vessel elements are similar to those of Trochodendron, whereas the fibers have no crossfield pit pairs but, rather, elliptical pits and sharp ends. Thus, both Trochodendron and Tetracentron are vessel bearing rather than vesselless, although their vessel elements are primitive.

The surface topography of the choroid plexus. Environmental, low and high vacuum scanning electron microscopy

Environmental scanning electron microscopy (ESEM) allows the examination of hydrated and dried specimens without a conductive metal coating which could be advantageous in the imaging of biological and medical objects. The aim of this study was to assess the performance and benefits of wet-mode and low vacuum ESEM in comparison to high vacuum scanning electron microscopy (SEM) using the choroid plexus of chicken embryos as a model, an organ of the brain involved in the formation of cerebrospinal fluid in vertebrates. Specimens were fixed with or without heavy metals and examined directly or after critical point drying with or without metal coating. For wet mode ESEM freshly excised specimens without any pre-treatment were also examined. Conventional high vacuum SEM revealed the characteristic morphology of the choroid plexus cells at a high resolution and served as reference. With low vacuum ESEM of dried but uncoated samples the structure appeared well preserved but charging was a problem. It could be reduced by a short beam dwell time and averaging of images or by using the backscattered electron detector instead of the gaseous secondary electron detector. However, resolution was lower than with conventional SEM. Wet mode imaging was only possible with tissue that had been stabilized by fixation. Not all surface details (e.g. microvilli) could be visualized and other structures, like the cilia, were deformed. In summary, ESEM is an additional option for the imaging of bio-medical samples but it is problematic with regard to resolution and sample stability during imaging.