Low-vacuum Scanning Electron Microscopy Research Articles

Cortical mechanics and myosin-II abnormalities associated with post-ovulatory aging: implications for functional defects in aged eggs.

Cellular aging of the egg following ovulation, also known as post-ovulatory aging, is associated with aberrant cortical mechanics and actomyosin cytoskeleton functions. Post-ovulatory aging is associated with dysfunction of non-muscle myosin-II, and pharmacologically induced myosin-II dysfunction produces some of the same deficiencies observed in aged eggs. Reproductive success is reduced with delayed fertilization and when copulation or insemination occurs at increased times after ovulation. Post-ovulatory aged eggs have several abnormalities in the plasma membrane and cortex, including reduced egg membrane receptivity to sperm, aberrant sperm-induced cortical remodeling and formation of fertilization cones at the site of sperm entry, and reduced ability to establish a membrane block to prevent polyspermic fertilization. Ovulated mouse eggs were collected at 21-22 h post-human chorionic gonadotrophin (hCG) (aged eggs) or at 13-14 h post-hCG (young eggs), or young eggs were treated with the myosin light chain kinase (MLCK) inhibitor ML-7, to test the hypothesis that disruption of myosin-II function could mimic some of the effects of post-ovulatory aging. Eggs were subjected to various analyses. Cytoskeletal proteins in eggs and parthenogenesis were assessed using fluorescence microscopy, with further analysis of cytoskeletal proteins in immunoblotting experiments. Cortical tension was measured through micropipette aspiration assays. Egg membrane receptivity to sperm was assessed in in vitro fertilization (IVF) assays. Membrane topography was examined by low-vacuum scanning electron microscopy (SEM). Aged eggs have decreased levels and abnormal localizations of phosphorylated myosin-II regulatory light chain (pMRLC; P = 0.0062). Cortical tension, which is mediated in part by myosin-II, is reduced in aged mouse eggs when compared with young eggs, by ∼40% in the cortical region where the metaphase II spindle is sequestered and by ∼50% in the domain to which sperm bind and fuse (P < 0.0001). Aging-associated parthenogenesis is partly rescued by treating eggs with a zinc ionophore (P = 0.003), as is parthenogenesis induced by inhibition of mitogen-activated kinase (MAPK) 3/1 [also known as extracellular signal-regulated kinase (ERK)1/2] or MLCK. Inhibition of MLCK with ML-7 also results in effects that mimic those of post-ovulatory aging: fertilized ML-7-treated eggs show both impaired fertilization and increased extents of polyspermy, and ML-7-treated young eggs have several membrane abnormalities that are shared by post-ovulatory aged eggs. These studies were done with mouse oocytes, and it remains to be fully determined how these findings from mouse oocytes would compare with other species. For studies using methods not amenable to analysis of large sample sizes and data are limited to what images one can capture (e.g. SEM), data should be interpreted conservatively. These data provide insights into causes of reproductive failures at later post-copulatory times. Not applicable. This project was supported by R01 HD037696 and R01 HD045671 from the NIH to J.P.E. Cortical tension studies were supported by R01 GM66817 to D.N.R. The authors declare there are no financial conflicts of interest.

The maintenance of inserted titanium implants: in-vitro evaluation of exposed surfaces cleaned with three different instruments.

Changes to titanium implants smooth-surfaces after instrumentation were comparatively analyzed using low-vacuum scanning electron microscopy (LV-SEM) and white-light confocal (WLC) profilometry, to accurately evaluate curved surfaces. Sixty titanium implants screwed to their abutments were randomly split into three groups for cleaning treatment with (S) stainless-steel Gracey-curettes, (T) titanium Langer-curettes, and (P) an ultrasonic-device with the probe covered with a plastic-tip. One sector of each implant was left unprocessed (U). The other sectors were cleaned for either 60s, to simulate a single cleaning session, or 180s to simulate a series of sessions. Surface morphology was analyzed by LV-SEM, without metal sputtering. Quantitative evaluations of the roughness of surfaces were performed using a WLC-profilometer. The Wilcoxon and the Mann-Whitney tests were used in statistical comparisons. U-surfaces showed that thin transverse ridges and grooves, i.e. a polarized surface roughness was substantially compromised after S-instrumentation. Small surface alterations, increasing with time, were also recorded after T-·and·P-instrumentation, although to a lesser degree. The gap of the fixture-abutment connection appeared almost completely clean after T-, clotted with titanium debris after S-, and clotted with plastic debris after P-treatment. The mean roughness (Ra) was unchanged after P-, significantly increased after S- and decreased after T-treatment, when compared with U. The Rz roughness-parameter, calculated along the fixture Y-axis, of S, T, and P resulted similar and significantly lower than that of U. Rz (X-axis) resulted unchanged after P-, slightly increased (+40%) after T-, and greatly increased (+260%) after S-treatment, this latter being statistically significant when compared with U. The careful use of titanium-curettes could produce only minimal smooth surface alteration particularly over prolonged treatments, and avoid debris production that could endanger implant preservation.

SCANNING ELECTRON MICROSCOPY OF THE RAT ADRENAL GLAND AFTER SURGICAL LASER EXPOSURE

Aim. We studied via low vacuum scanning electron microscopy the effects of a surgical laser exposure to adrenal glands and results of regeneration processes after. Materials and methods.Purpose of this work is modeling of effects of the removal with a surgical laser a pathological focus in the adrenal glands. For this Wistar male rat (n = 19) adrenal glands were researched without the laser exposure, immediately after it and 1 month later. Results . Immediately after exposure occurs laser ablation crater with rough edges and melted surface penetrated by equidistant pores, which are footprints of blood vessels. Beneath of the surface are numerous vaporizationbubbles. Around the crater, the surface wrinkles and sags due to decreased ability to retain water. 1 month after the laser damage, the affected area tightened by a scar. Its coarse bundles of collagen fibers braid shapeless lumps of coal and caverns. Tissues with normal appearance are close to the scar, both outside and inside of the organ. The wrinkling and the sagging are absent. The undamaged organ part has retained the previous shape, without hypertrophies. The damaged part has shrunk. The nature of the regeneration processes indicates a low probability of a relapse after the destruction of a pathological focus via the surgical laser exposure.

C4-P-10Three-dimensional ultrastructural analysis of secretory granules in histological paraffin sections of endocrine tumors using low-vacuum SEM

Secretory granules of endocrine tumors are traditionally identified by transmission electron microscopy of tissue samples. Recently, we developed a simple method for three-dimensional morphological analysis of conventional paraffin sections using low-vacuum scanning electron microscopy (SEM) [1], and showed that this technique was useful for pathological diagnosis of renal biopsy samples [2]. In this study, we investigated whether this method is useful as an adjunct in pathological diagnosis of endocrine tumors. Preparations were processed according to our original method. Paraffin sections (5 μm thick) obtained from a normal rat pancreas as a control, a human pituitary adenoma and a colorectal carcinoid tumor were generated by the conventional method. After deparaffinization, the sections were stained only with platinum blue aqueous solution (TI-blue staining kit: Nisshin EM, Tokyo) for 15 min at room temperature, and then observed by low-vacuum SEM (TM-1000, TM3030 plus: Hitachi, Tokyo) at high magnification (~30,000×). Under low-vacuum SEM, secretory granules of each cell could be identified as bright spherical granules in each paraffin-sectioned tissue, depending on the contrast of BSE signals. Figure shows low-vacuum SEM image (a) and TEM image (b) of endocrine secretory granules 100–260nm in diameter (arrows) in colorectal carcinoid tumor cells. Our results suggest that low-vacuum SEM of conventional paraffin sections is suitable for the pathological diagnosis of endocrine tumors. [1] Inaga et al., Arch. Histol. Cytol., 72 (2009) 101-106. [2] Okada et al., Biomed. Res., 35 (2014) 345-350. C4-P-09 doi:10.1093/jmicro/dfv327

Evaluation of short-term ex vivo culture with air/liquid system of porcine cornea

Regarding therapies for treatment of corneal wounds, ex vivo corneal culture is the most effective for minimizing expensive animal studies. Eighteen porcine enucleated eyes were soaked in 0.2% povidone iodine solution for disinfection prior to cornea excision. Subsequently, corneas were excised from whole eyes and filled with an agar/medium mixture. Corneas were transferred into culture dishes, after which culture medium was added until the limbus was covered. Cultures were then placed onto a plate rocker to mimic blinking action, followed by incubation at 37°C and 5% CO2. Corneas were harvested on Days 0, 3, and 7 after incubation, and optical coherence tomography (OCT) was performed on Day 7. Two eyes from each group were fixed in 2% glutaraldehyde/4% paraformaldehyde for low-vacuum scanning electron microscopy (LV-SEM), and four eyes from each group were fixed in 10% neutral-buffered formalin for histological analysis. OCT results showed that central corneal thickness significantly increased by Day 7 compared to Day 0 (p&lt;0.05). Using LV-SEM, gaps between endothelial cells were detected on Day 7 of ex vivo culture. In the histological evaluation, four to five stratified squamous cell layers, wing cells, and basal cells in the epithelium as well as flat-shaped keratocytes in the stroma were found on Day 0. By Day 7, stratified squamous cells and basal cells had decreased in number, and slightly round-shaped keratocytes were observed; however, the number of keratocytes was similar to that on Day 0. In this short-term ex vivo culture, epithelium and endothelium were sensitive to culture, whereas stroma and keratocytes were well maintained. An additional deswelling method will be needed to obtain more successful results in porcine corneal ex vivo culture.

First evidence of heat treatment during the early Neolithic in northeastern Italy

The site of La Vela sector VII (Trentino-Alto Adige – Italy), provides one of the few complete stratigraphic sequences, ranging from the Mesolithic to the middle Neolithic, in northwestern Italy. The site is located in the Adige valley, a way of communication between the Po plan and the Alps. This research focuses on the early Neolithic lithics ascribable to the Gaban facies, dated to 5000–4700 cal BC. Different rocks, coming from local exposures, have been exploited. The goal of the reduction sequence was the production of blades by pressure knapping technique. The technological study of the assemblage raised a particular interest for the presence of flint artifacts suggesting the usage of controlled heat treatment. The presence of this technique has been tested by an experimental approach based on Fourier-Transform infrared (FT-IR) spectroscopy. Complementary microstructural and analytical characterizations with low-vacuum scanning electron microscopy (LVSEM) observations combined with energy dispersive X-ray spectroscopy (EDXS) have been performed. The analyses confirm the presence of artifacts intentionally heat treated. The thermal treatment, achieved in order to increase the quality of the blade production, is the first evidence of this technique in northeastern Italy during the early Neolithic.

Applications of scanning electron microscopy in earth sciences

This paper expounds upon the basic principle of scanning electron microscopy (SEM), the main features of image types, and different signals, and the applications and prospects in earth sciences research are reviewed. High-resolution field emission SEM allows observation and investigation of a very fine micro area in situ. Using low-vacuum mode SEM, geological insulating samples can be analyzed directly without coating, demonstrating the wide application prospect. Combined with backscatter detector (BSE), energy dispersal X-ray spectroscopy (EDS), cathodoluminescence spectrometry (CL), and electron back-scattering diffraction (EBSD), SEM can yield multiple types of information about geological samples at the same time, such as superficial microstructure, CL analysis, BSE image, component analysis, and crystal structure features. In this paper, we use examples to discuss the geological application of SEM. We stress that we should not only focus on the CL image analysis, but strengthen CL spectrum analyses of minerals. These results will effectively reveal the mineral crystal lattice defects and trace element composition and can help to reconstruct mineral growth conditions precisely.

A Scanning Electron Microscope Study and Statistical Analysis of Adipocyte Morphology in Lipofilling

The aim of this study was to evaluate the effects on adipocyte morphology of 2 techniques of fat harvesting and of fat purification in lipofilling, considering that the number of viable healthy adipocytes is important in fat survival in recipient areas of lipofilling. Fat harvesting was performed in 10 female patients from flanks, on one side with a 2-mm Coleman cannula and on the other side with a 3-mm Mercedes cannula. Thirty milliliter of fat tissue from each side was collected and divided into three 10 mL syringes: A, B, and C. The fat inside syringe A was left untreated, the fat in syringe B underwent simple sedimentation, and the fat inside syringe C underwent centrifugation at 3000 rpm for 3 minutes. Each fat graft specimen was processed for examination under low-vacuum scanning electron microscope. Diameter (μ) and number of adipocytes per square millimeter and number of altered adipocytes per square millimeter were evaluated. Untreated specimens harvested with the 2 different techniques were first compared, then sedimented versus centrifuged specimens harvested with the same technique were compared. Statistical analysis was performed using Wilcoxon signed rank test. The number of adipocytes per square millimeter was statistically higher in specimens harvested with the 3-mm Mercedes cannula (P = 0.0310). The number of altered cells was statistically higher in centrifuged specimens than in sedimented ones using both methods of fat harvesting (P = 0.0080) with a 2-mm Coleman cannula and (P = 0.0050) with a 3-mm Mercedes cannula. Alterations in adipocyte morphology consisted in wrinkling of the membrane, opening of pore with leakage of oily material, reduction of cellular diameter, and total collapse of the cellular membrane. Fat harvesting by a 3-mm cannula results in a higher number of adipocytes and centrifugation of the harvested fat results in a higher number of morphologic altered cells than sedimentation.

Crystal orientation measurements using SEM–EBSD under unconventional conditions

Electron backscatter diffraction (EBSD) is a micro-analytical technique typically attached to a scanning electron microscope (SEM). The vast majority of EBSD measurements is applied to planar and polished surfaces of polycrystalline bulk specimen. In this paper, we present examples of using EBSD and energy-dispersive X-ray spectroscopy (EDX) to analyze specimens that are not flat, not planar, or not bulk – but pillars, needles, and rods. The benefits of low vacuum SEM operation to reduced drift problems are displayed. It is further demonstrated that small and thin specimens enhance the attainable spatial resolution for orientation mapping (by EBSD or transmission Kikuchi diffraction) as well as for element mapping (by EDX).

Marginal Adaptation Evaluation of Biodentine and MTA Plus in "Open Sandwich" Class II Restorations.

This study aimed at evaluation of two different commercially available calcium silicate materials (Biodentine and mineral trioxide aggregate [MTA] Plus) used as dentin substitute. Sixty Class II cavities were prepared in extracted mandibular third molars, with margins extending 1 mm below the cementum-enamel junction. The samples were divided into three groups on the basis of dentin substitute used: resin modified glass ionomer cement, Biodentine, and MTA Plus. Cavities were restored with composite resins in an "open sandwich" technique. The samples were subjected to alternate aging in phosphate buffered saline and cyclic loading. Marginal adaptation was evaluated in terms of "continuous margin" at the gingival margin, using a low vacuum scanning electron microscope. Statistical analysis was done with two-way analysis of variance with Holm-Sidak's correction for multiple comparisons. The glass ionomer group and Biodentine group presented an overall 83% and 91% of continuous margins, with no difference between them. MTA Plus showed least values of continuous margins. Granular deposits were seen over the surface of Biodentine and MTA Plus. Biodentine and resin-modified glass ionomer cement, when used as a dentin substitute under composite restorations in open sandwich technique, gave satisfactory marginal adaptation values. Contemporary calcium silicate materials can be used as dentin substitute materials in "open sandwich" Class II restorations. This study evaluates the marginal adaptation of Biodentine, MTA Plus, and resin modified glass ionomer cement used as dentin substitutes and reports better adaptation obtained with Biodentine and glass ionomer cement.

Studying the surface layers of products using a low-vacuum scanning electron microscope

The results of studying parameters of the surface layer after the tribological testing of different materials using a low-vacuum Phenom ProX scanning electron microscope are presented. It is shown that its use for the analysis of surface layers provides a very accurate estimate of their main parameters, i.e., volume topography and chemical composition for an insignificant (several seconds) sample setup time. The results of analysis of worn surfaces of FeGr1Cu19Sn0.9(MoS2)0.5 powder steels obtained by sintering and infiltration of a copper alloy, oxidized aluminum alloy AlCu4MgSi, and Vitalium Co-Cr-Mo alloy used for dental prostheses are presented as examples.

In vitro and in vivo removal of oral Candida from the denture base.

To clarify the effectiveness of ultrasonic cleaning for removing Candida lodged in the denture base. In vitro - Specimens of acrylic resin for denture plates contaminated with Candida cells were ultrasonically cleaned for 30 min. Washings were sampled every 5 min and cultured to investigate residual contamination, measured as colony forming units/ml, and the surfaces of the specimens were subjected to low-vacuum scanning electron microscopy (LV-SEM). In vivo - A total of 24 maxillary denture bases were brushed for 2 min under running tap water, then ultrasonically cleaned for 30 min. The washings were sampled every 5 min and cultured to investigate residual contamination. In vitro - Maximum removal was achieved during the first 5 min of cleaning, with the mean CFU/ml counts significantly declining over time. More than 85% of all Candida was removed within the first 15 min in specimens inoculated with phosphate-buffered saline suspensions of Candida albicans and Candida glabrata. LV-SEM revealed a decreased number of Candida on the surface of the specimens after 30 min of ultrasonic cleaning. In vivo - Maximum removal was achieved during the first 5 min of cleaning, then the mean CFU/ml count significantly declined during the first 10 min. Ultrasonic cleaning for 15 min removed 88.4% of Candida species from the denture base. Ultrasonic cleaning is a reliable and simple method for removing Candida lodged in the denture base.

Investigation of the effects of cadmium by micro analytical methods on Lycopersicon esculentum Mill. roots.

The interactions between cadmium stress and plant nutritional elements have been investigated on complete plant or at the level of organs. This study was undertaken to contribute to the exploration of the physiological basis of cadmium phytotoxicity. We examined the changes in the nutritional element compositions of the root epidermal cells of the seedlings of Lycopersicon esculentum Mill. at the initial growth stages that is known as the most sensitive stage to the stress. Effects of cadmium stress on the seedlings of Lycopersicon esculentum Mill. were examined by EDX (Energy Dispersive X-Ray Microanalysis) assay performed with using low vacuum (∼ 24 Pascal) Scanning Electron Microscopy. In the analysis performed at the level of root epidermal cells, some of the macro- and micronutrient contents of the cells (carbon, oxygen, nitrogen, phosphorus, potassium, calcium, magnesium, sulfur, iron, copper, and zinc levels) were found to change when the applying toxic concentrations of cadmium. There was no change in the manganese and sodium content of the epidermal cells. It was concluded that the changes in nutritional element composition of the cells can be considered as an effective parameter in explaining the physiological mechanisms of cadmium-induced growth inhibition.

Crystallographic characterization of eggshells as scaffolds for bone regeneration

The aim of this work was to characterize six different eggshell types as future osseous regeneration scaffolds. The study design was cross-sectional observational. The study was conducted in the Laboratory of Materials and the chemical analysis in the Faculty of Chemistry, UNAM, Mexico. Fifty samples of fresh eggs were obtained, 10 from each species (hen, turkey, duck, dove and quail). Square-shaped pieces of about 2 x 2 cm were obtained from the eggshells by means of a high-speed handpiece with a # 701 carbide bur. From the total of eggshell pieces, the ones coming from fresh eggs were included, and the ones showing signs of fracture or spots were excluded. Their morphology was analyzed with a low vacuum scanning electron microscope (JEOL 5200), with an accelerating voltage of 20 kV. The chemical elemental analysis was performed with an EDAX energy dispersive spectrometer (EDS) and the chemical composition was determined by means of a Siemens diffractometer. All of the eggshells were constituted by calcium carbonates, and their outer structure was less porous than the inner one, sometimes with granules and cuticles firmly attached to the inner surface of the eggshell. The eggshell major chemical components were mainly: oxygen, calcium, phosphorous, magnesium, sulfur and carbon. There are no significant quantities of sulfur (S) in the turkey and quail eggshells. These kinds of eggshells have not been tested yet as a scaffold, but it is expected that they may be used soon in bone regeneration once the organic part is removed. Eggshells are largely waste material. The authors anknowledge the technical support to Chem. Rafael Ivan Puente Lee

Glomerular basement membrane injuries in IgA nephropathy evaluated by double immunostaining for α5(IV) and α2(IV) chains of type IV collagen and low-vacuum scanning electron microscopy.

The glomerulus contains well-developed capillaries, which are at risk of injury due to high hydrostatic pressure, hyperfiltration, hypertension and inflammation. However, the pathological alterations of the injured glomerular basement membrane (GBM), the main component of the glomerular filtration barrier, are still uncertain in cases of glomerulonephritis. We examined the alterations of the GBM in 50 renal biopsy cases with IgA nephropathy (31.8 ± 17.6 years old) using double immunostaining for the α2(IV) and α5(IV) chains of type IV collagen, and examining the ultrastructural alterations by transmission electron microscopy (TEM) and low-vacuum scanning electron microscopy (LV-SEM). The GBM of IgA nephropathy cases showed various morphological and qualitative alterations. In the TEM findings, thinning, gaps, rupture, thickening with a lamellar and reticular structure and double contours were detected in the GBM. Double immunostaining for α5(IV) and α2(IV) showed thickening of the GBM with reduced α5(IV) and increased α2(IV), or mosaic images of α5(IV) and α2(IV), and holes, fractures, spiny projections and rupture of α5(IV) in the GBM. In addition, LV-SEM showed an etched image and multiple holes in a widening and wavy GBM. These findings might be associated with the development of a brittle GBM in IgA nephropathy. Glomerular basement membrane alterations were frequently noted in IgA nephropathy, and were easily evaluated by double immunostaining for α2(IV) and α5(IV) of type IV collagen and LV-SEM. The application of these analyses to human renal biopsy specimens may enhance our understanding of the alterations of the GBM that occur in human glomerular diseases.

Raman-in-SEM, a multimodal and multiscale analytical tool: Performance for materials and expertise

The availability of Raman spectroscopy in a powerful analytical scanning electron microscope (SEM) allows morphological, elemental, chemical, physical and electronic analysis without moving the sample between instruments. This paper documents the metrological performance of the SEMSCA commercial Raman interface operated in a low vacuum SEM. It provides multiscale and multimodal analyses as Raman/EDS, Raman/cathodoluminescence or Raman/STEM (STEM: scanning transmission electron microscopy) as well as Raman spectroscopy on nanomaterials. Since Raman spectroscopy in a SEM can be influenced by several SEM-related phenomena, this paper firstly presents a comparison of this new tool with a conventional micro-Raman spectrometer. Then, some possible artefacts are documented, which are due to the impact of electron beam-induced contamination or cathodoluminescence contribution to the Raman spectra, especially with geological samples. These effects are easily overcome by changing or adapting the Raman spectrometer and the SEM settings and methodology. The deletion of the adverse effect of cathodoluminescence is solved by using a SEM beam shutter during Raman acquisition. In contrast, this interface provides the ability to record the cathodoluminescence (CL) spectrum of a phase. In a second part, this study highlights the interest and efficiency of the coupling in characterizing micrometric phases at the same point. This multimodal approach is illustrated with various issues encountered in geosciences.

Prehistorical Pediculus humanus capitis infestation: quantitative data and low vacuum scanning microscopy.

A pre-Columbian Peruvian scalp was examined decades ago by a researcher from the Oswaldo Cruz Foundation. Professor Olympio da Fonseca Filho described nits and adult lice attached to hair shafts and commented about the origin of head lice infestations on mankind. This same scalp was sent to our laboratory and is the subject of the present paper. Analysis showed a massive infestation with nine eggs/cm2 and an impressive number of very well preserved adult lice. The infestation age was roughly estimated as nine months before death based on the distance of nits from the hair root and the medium rate of hair growth. A small traditional textile was associated with the scalp, possibly part of the funerary belongings. Other morphological aspects visualized by low-vacuum scanning electron microscopy are also presented here for adults and nits.

Effect of flowable composite liner and glass ionomer liner on class II gingival marginal adaptation of direct composite restorations with different bonding strategies

ObjectivesThe purpose of the present study was to comparatively evaluate the effect of flowable composite resin liner and resin modified glass ionomer liner on gingival marginal adaptation of class II cavities restored using three bonding agents (Single Bond 3M ESPE, One Coat Self Etching Bond Coltene Whaledent; Adper Easy Bond Self-Etch Adhesive 3M ESPE) and respective composite resins, under cyclic loading. The marginal adaptation was evaluated in terms of ‘continuous margin’ (CM) at the gingival margin. Materials and methodsNinety class II cavities with margins extending 1mm below the cement–enamel junction were prepared in extracted mandibular third molars. The samples were divided into three groups: no liner placement; 0.5–1mm thick flowable resin liner placement (Filtek Z350 XT flowable resin) on gingival floor and; light cure glass ionomer (Ketac N100) liner. The groups were further subdivided into three sub-groups on the basis of the bonding agents used. Cavities were restored with composite resins (Z350 for Single Bond and Adper Easy Bond; and Synergy D6 Universal, for One Coat Self Etching Bond) in 2mm increments and the samples were mechanically loaded (60N, 1,50,000 cycles). Marginal adaptation was evaluated using a low vacuum scanning electron microscope. Statistical analysis was done with two way ANOVA with Holm–Sidak's correction for multiple comparisons. ResultsPlacement of flowable composite liner significantly improved the CM values of Single Bond (78±11%) and One Coat Self Etching Bond (77±9%) compared with no liner group, but the values of CM of Adper Easy Bond were not improved (61±12%). Placement of glass ionomer liner significantly improved the values of CM in all the sub-groups (78±9%, 72±10% and 77±10% for Single Bond, One Coat Self Etching Bond & Adper Easy Bond respectively) compared with no liner group. ConclusionsPlacement of liners improved the values of ‘continuous margin’ in the gingival floor of the proximal cavities restored with composite resins using different bonding agent. Clinical relevancePlacement of flowable composite liner or glass ionomer liner will improve the marginal integrity of composite restorations using etch-and-rinse and two bottle-two step self etch adhesives. To improve the marginal integrity of a single bottle adhesive, glass ionomer liner should be applied.

Focus on photonics and biomedical optics

Focus on photonics and biomedical optics

Morphological diagnosis of Alport syndrome and thin basement membrane nephropathy by low vacuum scanning electron microscopy.

Alport syndrome (AS) and thin basement membrane nephropathy (TBMN) are genetic disorders caused by mutations of the type IV collagen genes COL4A3, COL4A4, and/or COL4A5. We here aimed to investigate the three-dimensional ultrastructure of the glomerular basement membrane (GBM) in order to introduce a novel method of diagnosing AS and TBMN. The subjects were 4 patients with AS and 6 patients with TBMN. Conventional renal biopsy paraffin sections from AS and TBMN patients were stained with periodic acid methenamine silver (PAM) and observed directly under low vacuum scanning electron microscopy (LVSEM). The PAM-positive GBMs were clearly visible under LVSEM through the overlying cellular components. The GBMs showed characteristic coarse meshwork appearances in AS, and thin and sheet-like appearances in TBMN. At the cut side view of the capillary wall, the GBMs in AS appeared as fibrous inclusions between a podocyte and an endothelial cell, while the GBMs in TBMN showed thin linear appearances. These different findings of GBMs between AS and TBMN were easily observed under LVSEM. Thus, we conclude that three-dimensional morphological evaluation by LVSEM using conventional renal biopsy paraffin sections will likely be useful for the diagnosis of AS and TBMN, including for retrospective investigations.