Low-vacuum Scanning Electron Microscopy Research Articles

Investigation of the interface of fungal mycelium composite building materials by means of low-vacuum scanning electron microscopy.

Low-vacuum scanning electron microscopy (low-vacuum SEM) is widely used for different applications, such as the investigation of noncoated specimen or the observation of biological materials, which are not stable to high vacuum. In this study, the combination of mineral building materials (concrete or clay plaster) with a biological composite (fungal mycelium composite) by using low-vacuum SEM was investigated. Fungal biotechnology is increasingly gaining prominence in addressing the challenges of sustainability transformation. The construction industry is one of the biggest contributors to the climate crises and, therefore, can highly profit from applications based on regenerative fungal materials. In this work, a fungal mycelium composite is used as alternative to conventional insulating materials like Styrofoam. However, to adapt bio-based products to the construction industry, investigations, optimisations and adaptations to existing solutions are needed. This paper examines the compatibility between fungal mycelium materials with mineral-based materials to demonstrate basic feasibility. For this purpose, fresh and hardened concrete specimens as well as clay plaster samples are combined with growing mycelium from the tinder fungus Fomes fomentarius. The contact zone between the mycelium composite and the mineral building materials is examined by scanning electron microscopy (SEM). The combination of these materials proves to be feasible in general. The use of hardened concrete or clay with living mycelium composite appears to be the favoured variant, as the hyphae can grow into the surface of the building material and thus a layered structure with a stable connection is formed. In order to work with the combination of low-density organic materials and higher-density inorganic materials simultaneously, low-vacuum SEM offers a suitable method to deliver results with reduced effort in preparation while maintaining high capture and magnification quality. Not only are image recordings possible with SE and BSE, but EDX measurements can also be carried out quickly without the influence of a coating. Depending on the signal used, as well as the magnification, image-recording strategies must be adapted. Especially when using SE, an image-integration method was used to reduce the build-up of point charges from the electron beam, which damages the mycelial hyphae. Additionally using different signals during image capture is recommended to confirm acquired information, avoiding misinterpretations.

Direct In- and Out-of-Plane Writing of Metals on Insulators by Electron-Beam-Enabled, Confined Electrodeposition with Submicrometer Feature Size.

Additive microfabrication processes based on localized electroplating enable the one-step deposition of micro-scale metal structures with outstanding performance, e.g., high electrical conductivity and mechanical strength. They are therefore evaluated as an exciting and enabling addition to the existing repertoire of microfabrication technologies. Yet, electrochemical processes are generally restricted to conductive or semiconductive substrates, precluding their application in the manufacturing of functional electric devices where direct deposition onto insulators is often required. Here, the direct, localized electrodeposition of copper on a variety of insulating substrates, namely Al2 O3 , glass and flexible polyethylene, is demonstrated, enabled by electron-beam-induced reduction in a highly confined liquid electrolyte reservoir. The nanometer-size of the electrolyte reservoir, fed by electrohydrodynamic ejection, enables a minimal feature size on the order of 200nm. The fact that the transient reservoir is established and stabilized by electrohydrodynamic ejection rather than specialized liquid cells can offer greater flexibility toward deposition on arbitrary substrate geometries and materials. Installed in a low-vacuum scanning electron microscope, the setup further allows for operando, nanoscale observation and analysis of the manufacturingprocess.

Familial focal segmental glomerulosclerosis with Alport-like glomerular basement changes caused by paired box protein 2 gene variant.

Paired box protein 2 (PAX2) gene variant causes renal coloboma syndrome (MIM#120330). Further, they are associated with focal segmental glomerulosclerosis and characterized by basement membrane changes similar to Alport syndrome.Herein, we report an 8-year-old boy who presented with proteinuria and decreased renal function. His paternal uncle has focal segmental glomerulosclerosis and renal failure, and his paternal grandmother has renal failure and is receiving peritoneal dialysis. Further, his father has stage 2 chronic kidney disease. At 3years of age, his serum creatinine-estimated glomerular filtration rate was 40-50mL/min/1.73 m2. At 8years of age, his renal function further decreased and he had proteinuria (urinary protein/Cr 3.39g/g Cr). Renal histopathology showed oligonephronia and focal segmental glomerulosclerosis. A partial basket-weave pattern, similar to Alport syndrome, was also observed on a transmission electron microscope, and low-vacuum scanning electron microscopy revealed coarse meshwork changes in the glomerular basement membrane. Genetic analysis revealed a PAX2 heterozygous variant (NM_003987.4:c.959C > G), a nonsense variant in which the serine at position 320 changes to a stop codon, in our patient and his father. PAX2 is a transcription factor that is important for the podocyte variant. However, podocytes with PAX2 gene variants may cause abnormal basement membrane production and repair, thereby resulting in Alport-like changes.

Pericyte detachment and renal congestion involve interstitial injury and fibrosis in Dahl salt-sensitive rats and humans with heart failure

Congestive heart failure produces fluid volume overload, central and renal venous pressure elevation, and consequently renal congestion, which results in worsening renal function. Pericyte detachment and pericyte-myofibroblast transition (PMT) were linked to renal interstitial fibrosis. Dahl salt-sensitive hypertensive (DahlS) rats are a non-surgical renal congestion model. The relation, however, between renal interstitial damage, pericyte morphology, and PMT in the renal congestion of DahlS rats has not been reported. DahlS rats (8-week-old) were fed normal salt (NS, 0.4% NaCl) or high salt (HS, 4% NaCl), and the left kidney was decapsulated to reduce renal interstitial hydrostatic pressure (RIHP) at 9 weeks old. One week after capsulotomy, both kidneys were analyzed by molecular and histological techniques. Renal pericyte structure was assessed in the body donors with/without venous stasis. Markers of tubulointerstitial damage, interstitial fibrosis, and PMT were upregulated in the right non-decapsulated kidney of DahlS rats fed HS. Renal tubular injury and fibrosis were detected in the HS diet groups in histological analysis. Pericyte detachment was observed in the right non-decapsulated kidney of DahlS rats fed HS by low vacuum-scanning electron microscopy. Decapsulation in DahlS rats fed HS attenuated these findings. Also, renal pericytes detached from the vascular wall in patients with heart failure. These results suggest that pericyte detachment and PMT induced by increased RIHP are responsible for tubulointerstitial injury and fibrosis in DahlS rats and humans with renal congestion. Renal venous congestion and subsequent physiological changes could be therapeutic targets for renal damage in cardiorenal syndrome.

Three-dimensional ultrastructure of the human placenta by informative low-vacuum scanning electron microscopy

Three-dimensional ultrastructure of the human placenta by informative low-vacuum scanning electron microscopy

Micromorphological observation of HLE cells under knockdown of Fascin using LV-SEM.

Liver cancer is one of the most prevalent cancers in Japan with hepatocellular carcinoma (HCC) as the major histological subtype. Successful novel treatments for HCC have been reported; however, recurrences or metastasis may occur, which results in poor prognoses and high mortality of HCC patients. Fascin, an actin-bundling protein, regulates cell adhesion, migration, and invasion. Its overexpression positively correlates with poor prognosis of malignant tumors, and Fascin is considered as one of the tumor biomarkers and therapeutic target proteins. In this study, we attempted to reveal the relationship between Fascin and HCC using HLE, one of the human HCC cell lines. We performed the study with classical immunocytochemistry and recently developed techniques, such as wound-healing assay, spheroid cultivation, and low-vacuum scanning electron microscopy (LV-SEM). Non-Fascin-knockdown (FKD) cell spheroid had a regular spherical appearance with tight cell-cell connections, while FKD cell spheroid had an irregular shape with loose cell-cell connections. Cells of non-FKD spheroid presented fibrous protrusions on the cell surface, contrarily, cells of FKD spheroids showed bulbous-shaped protrusions. Morphological observation of FKD and non-FKD HLE spheroids were performed using LV-SEM. Our study may help to reveal the roles of Fascin in the process of HCC formation and its malignancy.

Direct observation of epoxy resin blocks for renal biopsy by low-vacuum scanning electron microscopy

To improve the resolution of low-vacuum scanning electron microscopy (LVSEM), the epoxy resin block for the transmission electron microscopy (TEM) was observed directly with LVSEM. After observing ultrathin sections from renal biopsies of IgA nephropathy, membranous nephropathy, lupus nephritis, diabetic nephropathy (DM), thin basement membrane disease (TBMD), Alport’s syndrome, Fabry’s disease, and renal amyloidosis, the epoxy resin blocks of the same sites were observed by LVSEM and compared. The LVSEM image of the epoxy resin block corresponds to the negative of the TEM image, and when the gradation is reversed, the LVSEM image was comparable to the TEM image. At a low magnification of 100 ×, the entire specimen, including the glomerulus, was obtained. LVSEM at 5000 × magnification was sufficient to identify paramesangial deposits in IgA nephropathy and subepithelial electron-dense deposits (EDD) and spikes in membranous nephropathy. Glomerular basement membrane thickening in DM and thinning in TBMD could be sufficiently diagnosed with LVSEM at 6000 ×. Accumulation of ceramide in Fabry's disease was easily identified, but amyloid fibril could not be identified by LVSEM. LVSEM of renal biopsy epoxy resin blocks can replace TEM up to moderate magnification.

Scanning electron microscopy evaluation of enamel surfaces using different air-polishing powders in the orthodontic setting: an invitro study.

The aim of this invitro study was to quantify and compare changes of the enamel surface caused by periodical use of different air-polishing powders during multibracket therapy. Bovine high-gloss polished enamel specimens were air-polished using an AIR-FLOW® Master Piezon with maximum powder and water settings. Each specimen was blasted with sodium bicarbonate (AIR-FLOW® Powder Classic, Electro Medical Systems, Munich, Germany) and erythritol (AIR-FLOW® Powder Plus, Electro Medical Systems). Blasting duration was adapted to the powders' cleaning efficacy and corresponded to 25air-polishing treatments in apatient with braces. Aspindle apparatus ensured uniform guidance at adistance of 4 mm and a90° angle. Qualitative and quantitative assessments were performed with the use of low vacuum scanning electron microscopy. Following external filtering and image processing, arithmetical square height (Sa) and root mean square height (Sq) were determined. Both prophy powders caused asignificant increase in enamel roughness. Surfaces blasted with sodium bicarbonate (Sa = 64.35 ± 36.65 nm; Sq = 80.14 ± 44.80 nm) showed significantly (p < 0.001) higher roughness than samples treated with erythritol (Sa = 24.40 ± 7.42 nm; Sq = 30.86 ± 9.30 nm). The observed defects in enamel structure caused by sodium bicarbonate extended across prism boundaries. Prism structure remained intact after air-polishing with erythritol. Both applied air-polishing powders led to surface alterations. Despite shorter treatment times, sodium bicarbonate was significantly more abrasive than erythritol. Clinicians must compromise between saving time and abrasively removing healthy enamel.

Super-Stretchable Resistive Strain Sensor Based on Ecoflex–Gold Nanocomposites

Superstretchable resistive strain sensors can reversibly undergo severe deformations in response to different mechanical stimuli, leading to a variation in their ohmic resistance. They are finding an increasing number of applications in wearable electronics, biomedical devices, and soft robotics. The development of fabrication process of reliable, highly sensitive, and biocompatible superstretchable strain sensors remains a challenge, especially in view of their integration with medical devices. Here, we demonstrate the fabrication of a high-strain resistive sensor based on Ecoflex elastomeric films and conductive Au clusters, obtained by supersonic cluster beam deposition. An extensive characterization of the electromechanical behavior of the sensor shows stable operation up to 4500 working cycles, with a maximum strain of 500% and a gauge factor of 124. Different mechanical stimuli, such as elongation and indentation, can be detected, and the easy coupling of the sensor with an inflatable balloon-type urinary catheter is demonstrated. Aiming at shedding light on the observed electromechanical mechanisms, the nanostructured morphology of the metal–polymer hybrid layer was investigated by low-vacuum scanning electron microscopy.

Three-dimensional fine structures in deep fascia revealed by combined use of cryo-fixed histochemistry and low-vacuum scanning microscopy

Recent physiological studies have shown that the deep fascia has received much attention concerning clinical medicine; however, histological examination of the deep fascia has not been well established. In this study, we aimed to clarify and visualize the structure of the deep fascia by taking advantage of cryofixation techniques and low-vacuum scanning electron microscopy. As a result, the ultrastructural observations revealed three-dimensional stratification of the deep fascia composed of three layers: the first superficial layer consisting of collagen fibers extending in various directions with blood vessels and peripheral nerves; the second intermediate layer formed by single straight and thick collagen fibers with flexibility; and the third deepest layer, consisting of relatively straight and thin collagen fibers. We explored the use of two hooks to hold a piece of deep fascia in place through the course of cryo-fixation. A comparative observation with or without the hook-holding procedure would indicate the morphological adaptation to physiological stretch and contraction of the deep fascia. The present morphological approach paves the way to visualize three-dimensional ultrastructures for future biomedical studies including clinical pathophysiology.

A Novel Multi-Observation System to Study the Effects of Anterior Ocular Inflammation in Zinn's Zonule Using One Specimen.

Zinn's zonule is a fragile and thin tissue, and little is known about its pathogenesis. The aim of this study was to develop an experimental setup for a comprehensive analysis of Zinn's zonule. Rats were divided into two groups: a control group (n = 4) and an alkali injury group (n = 4). Seven days after injury, the eyes were enucleated, the anterior eye was dissected and embedded in gelatin, and macroscopic observations were made. The gelatin specimens were then embedded in paraffin and observed in detail by low-vacuum scanning electron microscopy, immunofluorescence, and quantitative reverse transcription polymerase chain reaction (RT-qPCR). The results show qualitative changes in Zinn's zonules in both macroscopic and microscopic observations. In addition, macrophage infiltration and increased matrix metalloproteinase 2 (MMP2) expression were observed in the injured group, consistent with the RT-qPCR results. The experimental system in this study allowed us to capture the morphological and molecular biological changes of Zinn's zonule and to gain insight into its pathogenesis. In conclusion, this study presents a new experimental setup for the comprehensive analysis of the rat Zinn's zonule. The results suggest that this system can be used in the future to study and analyze a variety of paraffin-embedded tissues and specimens.

Impact of radiotherapy on the morphological and compositional structure of intra-radicular dentin

Considering the side effects in the oral cavity and dental structures of radiotherapy (RDT) for head and neck cancer, this study aimed to evaluate the effects of RDT on the root dentin concerning the obliteration of dentinal tubules, the inorganic composition of intra-radicular dentin, and the integrity of collagen fibers. Thirty human canines were selected from a biobank and randomly divided into two groups (n=15). The samples were sectioned buccolingually, and a hemisection was used for structural analysis by scanning electron microscopy (SEM) and energy-dispersive X-ray spectrometer (EDS). Low-vacuum SEM images were obtained at 2000-x magnification to observe the obliteration of the dentinal tubules. Moreover, compositional evaluation was performed using EDS. After RDT, the SEM and EDS analyses were repeated using the same methodology. RDT was applied fractionally at 2 Gy per day, 5 days per week, for 7 weeks, resulting in a total dose of 70 Gy. The collagen integrity of the irradiated and non-irradiated samples was analyzed using Masson's trichrome and picrosirius red staining polarization microscopy. Samples subjected to RDT exhibited dentinal tubule obliteration (p < 0.001); low integrity of type I and III collagen fibers (p < 0.05); compositional reduction of calcium (p = 0.012), phosphorus (p = 0.001), and magnesium (p < 0.001); an increased Ca/P ratio (p < 0.001). RDT affects the structure of dentinal tubules, the inorganic composition of intra-radicular dentin, and the collagen fiber integrity in the root dentin, which may interfere with the effectiveness and durability of dental procedures.

PS-BPB10-6: THE RELATION BETWEEN PERICYTE DETACHMENT AND INTERSTITIAL FIBROSIS IN DAHL SALT-SENSITIVE HYPERTENSIVE (DAHLS) RATS

Background: Congestive heart failure produces the state of fluid volume overload, central venous pressure (CVP) elevation, and consequently renal venous pressure (RVP) elevation. We have recently developed a novel rat unilateral renal venous congestion model, in which ligation of the inferior vena cava between both renal veins artificially induced renal congestion in the left kidney only. In this model, pericyte detachment and pericyte-myofibroblast transition (PMT) around the vasa recta were involved in interstitial injury and fibrosis. Dahl salt-sensitive hypertensive (DahlS) rats on high salt diets exhibit hypertension, congestive heart failure, fluid volume overload, CVP and RVP elevation, and renal damage, considering to be a non-artificial renal congestion model. The relation, however, between renal interstitial injury and fibrosis, elevated RVP, and pericyte detachment in DahlS rats has not been reported. In the present study, we assessed the relation of pericyte detachment around the vasa recta and PMT with interstitial fibrosis in DahlS rats. Methods: Eight-week-old DahlS rats (Dahl/Mcwi) were divided into three groups: normal diet, 4% NaCl diet, and 8% NaCl diet groups, and their kidneys were analyzed after 2 weeks. Total mRNA and protein expression levels were quantified by real-time PCR and western blot, respectively. Tubulointerstitial damage and fibrosis were identified by histological analysis. Furthermore, we evaluated pericyte detachment around the vasa recta by low-vacuum scanning electron microscopy (LV-SEM). Results: The expression of KIM1, a tubulointerstitial damage marker; CNN1 and αSMA, interstitial fibrosis markers; and TAGLN, a smooth muscle protein22 and PMT marker; was significantly increased in a salt-dependent manner in DahlS rats. In histological analysis, hyperplasia of the extracellular matrix and renal tubular injury were detected in 4% and 8% NaCl diet groups. The positive area of PMT markers in the interstitial space around the vasa recta was also increased in the NaCl diet groups. Pericyte detachment from the vasa recta was observed in the NaCl diet groups by LV-SEM. Conclusions: Pericyte detachment and PMT are involved in tubulointerstitial injury and fibrosis of DahlS rats. Renal venous congestion and subsequent physiological changes could have a role in renal damage and fibrosis. Arterial hypertension has been emphasized for a long time in renal injury, and venous hypertension must also be focused on with the concept of renal congestion. Further understanding of this concept may lead to new therapeutic strategies for renal injury.

Imaging of Liver Tissues Using a Combination of Silver Impregnation and Low-Vacuum Scanning Electron Microscopy; A Simple Method for the High-Resolution Visualization of Reticulin Structures with Applicability to a Quantitative Analysis.

Low-vacuum scanning electron microscopy (LVSEM) enables the detailed three-dimensional imaging of archival tissues without special pretreatments. The clinical utility of LVSEM in the assessment of liver diseases has not yet been defined. So, we investigated the utility of LVSEM imaging in morphological assessments of normal and diseased liver tissues, with a focus on reticulin structures. Formalin-fixed tissue samples of two normal livers and two hepatocellular carcinomas with background regenerative nodules/areas were stained with platinum blue stain or silver-impregnated using Watanabe's method and then comparatively observed under LVSEM. We also evaluated the applicability of LVSEM imaging of liver tissues to a quantitative analysis using a digital image analysis technique. Optimal high-resolution images of reticulin structures were obtained using 10-μm-thick silver-impregnated sections. Reticulin fibers were clearly observed to run dendritically around sinusoids in normal livers, and markedly increased in regenerative nodules/areas. Normal reticulin frameworks were lost in hepatocellular carcinoma, leaving a few fragments of reticulin fibers within tumors. Moreover, when a quantitative analysis was applied to these images, we successfully demonstrated a significantly higher reticulin fiber density in regenerative nodules/areas than in the normal liver (P < 0.05). We not only obtained detailed three-dimensional images of reticulin structures in various liver tissues by LVSEM combined with silver impregnation but also showed their applicability to a quantitative analysis. The method presented herein may be applied to future studies for the more accurate diagnosis and better classification/risk stratification of various liver diseases.

Disulfiram Ophthalmic Solution Inhibited Macrophage Infiltration by Suppressing Macrophage Pseudopodia Formation in a Rat Corneal Alkali Burn Model

FROUNT is an intracellular protein that promotes pseudopodia formation by binding to the chemokine receptors CCR2 and CCR5 on macrophages. Recently, disulfiram (DSF), a drug treatment for alcoholism, was found to have FROUNT inhibitory activity. In this study, we investigated the effect of DSF eye drops in a rat corneal alkali burn model. After alkali burn, 0.5% DSF eye drops (DSF group) and vehicle eye drops (Vehicle group) were administered twice daily. Immunohistochemical observations and real-time reverse transcription-polymerase chain reaction (RT-PCR) analyses were performed at 6 h and 1, 4, and 7 days after alkali burn. Results showed a significant decrease in macrophage accumulation in the cornea in the DSF group, but no difference in neutrophils. RT-PCR showed decreased expression of macrophage-associated cytokines in the DSF group. Corneal scarring and neovascularization were also suppressed in the DSF group. Low-vacuum scanning electron microscopy imaging showed that macrophage length was significantly shorter in the DSF group, reflecting the reduced extension of pseudopodia. These results suggest that DSF inhibited macrophage infiltration by suppressing macrophage pseudopodia formation.

Evaluation of time exposure of different irrigation agents on radicular dentin erosion

Aim: The amount of root dentin erosion caused by Ethylene diamine tetraacetic acid (EDTA), Glycolic acid (GA), and Etidronic acid (HEDP) solutions applied with sonic activation (SA) for 1 and 3 minutes during the final irrigation was evaluated in this in vitro study. Methods: Using 5 ml of 2.5% NaOCl irrigation solution in each sample, 70 mandibular premolars were enlarged with the Reciproc R25 rotary instrument system. The samples were randomly divided according to the final irrigation protocol into 6 experimental groups (n:10) and 1 control group (n:10): Group 1   (17% EDTA – 1 min SA), Group 2   (17% EDTA – 3 min SA), Group 3   (10% GA – 1 min SA), Group 4   (10% GA – 3 min SA), Group 5   (18% HEDP – 1 min SA), Group 1   (18% HEDP – 3 min SA) and Group 7   (Negative control group-distilled water, 1 min SA). The teeth were divided into two parts longitudinally after the final irrigation protocol. A low vacuum scanning electron microscope (SEM) was used to image the samples’ coronal, middle, and apical root areas at x2000 magnification. Samples were evaluated using a triple-scoring system. Results: Data were analyzed by Shapiro Wilk, Kruskal Wallis H, Mann-Whitney U, and Dunn’s tests. There was no statistically significant difference between EDTA, GA, and HEDP in both periods (p &gt; 0.05). Conclusion: We saw similar amounts of erosion in dentin in 1 and 3 minutes of chelation applications for all chelators. Considering the smear removal efficiency of chelators, there is no harm in increasing the time in terms of erosion. There is a need for studies on the clinical applicability of chelation with a different duration time.   How to cite this article: Okumuş ÖF, Kaya S, Adıgüzel Ö, Yeniçeri Özata M. Evaluation of time exposure of different irrigation agents on radicular dentin erosion. J Med Dent Invest 2022;3:e221170. https://doi.org/10.5577/jomdi.e221170   Linguistic revision: The English in this manuscript has been checked by at least two professional editors, both native speakers of English.   Checked for plagiarism: Yes

Conductive cross-section preparation of non-conductive painting micro-samples for SEM analysis

Scanning electron microscopy (SEM) is a common method for the analysis of painting micro-samples. The high resolution of this technique offers precise surface analysis and can be coupled with an energy-dispersive spectrometer for the acquisition of the elemental composition. For light microscopy and SEM analysis, the painting micro-samples are commonly prepared as cross-sections, where the micro-sample positioned on the side is embedded in a resin. Therefore, the sequence of its layers is exposed after the cross-section is polished. In common cases outside of cultural heritage, a conductive layer is applied on the polished side, but in this field, the measurements are mostly done in low-vacuum SEM (LV-SEM). Although the charging effect is reduced in LV-SEM, it can still occur, and can hardly be prevented even with carbon tape or paint. This work presents two conductive cross-section preparation methods for non-conductive samples, which reduce charging effects without impairing the sample integrity.

Characterization of chemical elements of calcium silicate-based cements

The aim of this study was to characterize the chemical elements and surface of the cements: Biodentine®, Bio-C Sealer®, Endosequence® BC Sealer, MTA Angelus® and Portland Cement (PC). Material and methods: The cements were manipulated according to manufacturer’s recommendations, inserted in polyethylene tubes and kept in a 95% humidity incubator for 72 hours. The analysis of the chemical elements was performed by energy dispersive X-ray spectroscopy (EDS) coupled to a low vacuum Scanning Electron Microscope (SEM). The surface analysis was performed according to the distribution pattern, shape and particle size in images obtained using Field Emission Gun SEM (SEM-FEG). Data were analyzed descriptively for EDS chemical composition spectra and qualitatively for SEM-FEG images. Results: EDS analysis showed that all evaluated cements contained calcium and silicon rich particles. MTA Angelus® presented bismuth-rich particles, while Biodentine®, Bio-C Sealer® and Endosequence® presented zirconium-rich particles, the latter having tantalum in its composition. All materials showed a uniform distribution pattern of particles on the surface. Biodentine® and Endosequence® showed similarly shaped particles. Conclusion: The cements present particles with uniform distribution pattern and similar chemical composition, differentiating the presence of bismuth or zirconium.

A novel technique of STEM observation of TEM section using LVSEM and application of pathological diagnosis of renal biopsy.

The usefulness of the transmission electron microscope (TEM) for pathological diagnosis is apparent. However, high operating costs and other disadvantages have limited the ability to maintain and operate a TEM. In recent years, a general-purpose benchtop low-vacuum scanning electron microscope (LVSEM), which is inexpensive and easy to operate, has been developed and is expected to be applied in electron microscopic pathological diagnosis. To date, we have previously observed TEM ultrathin sections of Immunoglobulin A (IgA) nephropathy with a benchtop LVSEM using an ultra variable-pressure detector (UVD) and a newly developed holder for observing scanning transmission electron microscope (STEM) images (UVD-STEM holder) and compared the images with those obtained with typical TEM observations. We reported the results in the 53rd Annual Meeting of the Japanese Society for Clinical Molecular Morphology and the 64th Symposium of The Japanese Society of Microscopy and discussed the validity of the methods in the pathological diagnosis of IgA nephropathy and other renal diseases. As a result, we demonstrated the potential for pathological diagnosis using benchtop LVSEM. In this study, we similarly examined typical kidney diseases such as membranous nephropathy, lupus nephritis and amyloidosis. We could obtain sufficient data for the pathological diagnosis of IgA nephropathy, membranous nephropathy and lupus nephritis. However, it is difficult to detect amyloid fibres that are characteristic of amyloidosis. The development of this method is expected to expand the possibilities for pathological diagnosis using electron microscopy, including its application to other diseases.

Overexpression of Plasmalemmal Vesicle-Associated Protein-1 Reflects Glomerular Endothelial Injury in Cases of Proliferative Glomerulonephritis with Monoclonal IgG Deposits

Proliferative glomerulonephritis with monoclonal IgG deposits (PGNMID) occasionally presents refractory nephrotic syndrome resulting in poor renal prognosis, but its etiology is not fully elucidated. Given that glomerular endothelial cell (GEC) stress or damage may lead to podocytopathy and subsequent proteinuria, as in thrombotic microangiopathy (TMA), diabetic kidney disease, and focal segmental glomerulosclerosis, we investigated the evidence of glomerular endothelial injury by evaluating the expression of plasmalemmal vesicle-associated protein-1 (PV-1), a component of caveolae in the cases of PGNMID. We measured the immunofluorescent PV-1 intensities of 23 PGNMID cases and compared with those of primary membranoproliferative glomerulonephritis (MPGN) (n= 5) and IgA nephropathy (IgAN) (n= 54) cases. PV-1 localization was evaluated with Caveolin-1, and CD31 staining, and the ultrastructural analysis was performed using a low-vacuum scanning electron microscope (LVSEM). To check the association of podocyte injury, we also conducted 8-oxoguanine and Wilms tumor 1 (WT1) double stain. We then evaluated PV-1 expression in other glomerulitis and glomerulopathy such as lupus nephritis and minimal change disease. The intensity of glomerular PV-1 expression in PGNMID is significantly higher than that in the other glomerular diseases, although the intensity is not associated with clinical outcomes such as urinary protein levels or renal prognosis. Immunostaining and LVSEM analysis revealed that glomerular PV-1 expression is localized in GECs in PGNMID. 8-oxoguanine accumulation was detected in WT1-positive podocytes but not in PV-1-expressing GECs, suggesting GEC-derived podocyte injury in PGNMID. PV-1 overexpression reflects glomerular endothelial injury, which could be associated with podocyte oxidative stress in PGNMID cases.