Effect of processing methods for transmission electron microscopy on corneal collagen fibrils diameter and spacing

Introduction: The corneal tissue was processed in fixatives and embedded in resin for transmission electron microscopy to observe the ultrastructure of the collagen fibrils (CFs). The effect of these processing methods on the CF diameter and the interfibrillar spacing was studied. Methods: Four normal human corneal buttons were used for this study. A part of each cornea was fixed in 2.5% glutaraldehyde containing cuprolinic blue in sodium acetate buffer and embedded in spurr's resin (SpurrCB). A second part of each cornea was fixed in 2.5% glutaraldehyde + osmium tetroxide and embedded spurr's resin (SpurrOsm). The third part of each cornea was fixed in paraformaldehyde (4%) and embedded in LR White at 4°C (LRWhite). Ultrathin sections were stained with uranyl acetate and lead citrate. Results: In the tissue, fixed in SpurrCB, the diameter was 38.4 ± 5.9 nm and spacing between CF was 52.5 ± 5.3 nm. In the tissue fixed in SpurrOsm, the diameter was 28.37 ± 5.84 nm and spacing between CF was 45 ± 4.57 nm. In the tissue fixed in LR White, the CF diameter was 24 ± 2.3 nm and spacing between CF was 39.0 ± 4.2 nm. The diameters and interfibrillar spacing of the tissue processed by SpurrCB, SpurrOsm, and LRWhite were significantly different (P < 0.001) from one another. Conclusion: Our study shows that there is a variation in the CF diameter and spacing depending on the method of fixation and embedding resins used. This needs to be considered when comparative studies using different methods are done. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.     

Characterization of ultrastructure and collagen composition of the teratoma membrane: Comparison to the amniotic membrane

The structural and morphological properties of the teratoma membrane were investigated to better understand the pathogenesis of ovarian teratomas. A mature cystic teratoma and amnion were obtained from patients who underwent laparoscopic cystectomy and uncomplicated delivery, respectively. The teratoma membrane was divided into three layers according to the results of the histological analysis. Each layer showed distinct morphological properties, including an outer layer that was uniformly arranged, a middle layer with an irregular pattern of fibers, and an inner layer that was structurally dense with a wavy pattern of fibers. The morphology of the layers of the amniotic membrane was the reverse that of the teratoma membrane. In the teratoma membrane, the outer layer was primarily composed of type III collagen and the inner layer had a large amount of type III and IV collagen. The amniotic membrane showed a small amount of type III collagen in the outer layer, whereas the inner layer had large amounts of type I, III, and IV collagen. In the teratoma membrane, the collagen fibrils were arranged regularly in the outer layer, but irregularly in the inner layer. In the amniotic membrane, the arrangement of collagen fibrils was the reverse that of the teratoma membrane. Additionally, the collagen fibrils in the teratoma membrane were thinner than those of the amniotic membrane and had slightly shorter d‐spacing. Two membranes showed the differences in collagen fibril arrangement, which may caused by the different functional roles. Microsc. Res. Tech. 76:432–441, 2013. © 2013 Wiley Periodicals, Inc.     

Short‐term effect of cryotherapy on human scleral tissue by atomic force microscopy

This study investigated the inflammatory effect of cryotherapy application on collagen matrix network in human infant sclera. Donor scleral tissues taken from three infant patients divided into five groups: control group, sham‐treated group, and three cryotreated groups. In the cryotherapy groups, the sclera was treated for 5 s, 10 s, and 20 s with ?80°C freezing by a cryosurgical system. The cryotreated reactions were examined using double histological analysis with hematoxylin‐eosin and Masson's trichrome, and atomic force microscopy analysis to quantify the diameter and D‐banding of collagen fibrils. The infant scleral tissues treated with cryotherapy showed a significantly increased collagen density associated with inflammatory response (p < 0.05), increased fibril diameter (p < 0.005) compared to the scleral tissues in the control group. The results directly suggest that the cryotherapy affects the morphology of scleral collagen. SCANNING 35:302‐307, 2013. © 2012 Wiley Periodicals, Inc.     

Investigation of nanostructural changes following acute injury using atomic force microscopy in rabbit vocal folds

There continues to be a paucity of data regarding the nanostructural changes of vocal fold (VF) collagen after injury. The aim of this study is to investigate the nanostructural and morphological changes in the rabbit VF lamina propria following acute injury using atomic force microscopy (AFM). Unilateral VF injury was performed on 9 New Zealand breeder rabbits. Sacrifice and laryngeal harvest were performed at three time points: 1 day, 3 days, and 7 days after injury. Histology and immunohistochemistry data were collected to confirm extracellular matrix (ECM) changes in rabbit VF. The progressive changes in thickness and D‐spacing of VF collagen fibrils were investigated over a 7‐day postinjury period using AFM. At post‐injury day 1, a fibrin clot and inflammatory cell infiltration were observed at the injured VF. The inflammatory score at postinjury day 1 was highest in injured VF tissue, with a significant decrease at postinjury day 7. The immunoreactivity of inflammatory proteins (COX‐2, TNF‐α) was observed in VF up to day 7 after injury. AFM investigation showed clustered and disorganized collagen fibrils at the nanoscale resolution at post‐injury day 7. Collagen fibrils in injured VF at postinjury day 7 were significantly thicker than control and postinjury days 1 and 3 (P < 0.001). D‐spacing of collagen at postinjury day 7 was not studied due to loss of distinct edges resulting from immature collagen deposition. AFM investigation of VF could add valuable information to understanding micromechanical changes in VF scar tissue. Microsc. Res. Tech. 78:569–576, 2015. © 2015 Wiley Periodicals, Inc.     

Papain‐gel degrades intact nonmineralized type I collagen fibrils

Papain‐gel has been utilized as a chemo‐mechanical material for caries removal due to its ability to preserve underlying sound dentin. However, little is known about the effect of the papain enzyme on intact type I collagen fibrils that compose the dentin matrix. Here we sought to define structural changes that occur in intact type I collagen fibrils after an enzymatic treatment with a papain‐gel. Intact and nonmineralized type I collagen fibrils from rat tail were obtained and treated with a papain‐gel (Papacarie) for 30 s, rinsed with water and imaged using an atomic force microscope (AFM). Additionally, polished healthy dentin specimens were also treated using the same protocol described above and had their elastic modulus (E) and hardness (H) measured by means of AFM‐based nanoindentation. AFM images showed that the papain‐gel induced partial degradation of the fibrils surface, yet no rupture of fibrils was noticed. The distinction between gap and overlap zones of fibrils vanished in most regions after treatment, and overlap zones appeared to be generally more affected. Mechanical data suggested a gradual decrease in E and H after treatments. A significant two‐fold drop from the values of normal dentin (E=20±1.9, H=0.8±0.08 GPa) was found after four applications (E=9.7±3.2, H=0.24±0.1 GPa) (P<0.001), which may be attributed to the degradation of proteoglycans of the matrix. In summary, this study provided novel evidence that intact nonmineralized type I collagen fibrils are partially degraded by a papain‐gel. SCANNING 31: 253–258, 2009. © 2010 Wiley Periodicals, Inc.     

Postoperative effect of radiofrequency treatments on the rabbit dermal collagen fibrillary matrix

This study quantitatively examined the short and mid‐long term effects of radiofrequency (RF) treatment on the normal dermal collagen fibrils of live rabbits. Effects were evaluated by histology and scanning probe microscopy analysis of dermal tissues treated using three RF energy levels (10, 20, and 30 W) and either a single‐ or multiple‐pass procedure. Progressive changes in the morphology of rabbit dermal collagen fibrils were investigated over a 30‐day post‐treatment period. All RF‐treated groups, except for the low‐energy group (10 W), displayed more prominent inflammatory responses compared to the control. This inflammatory response was more prominent a day after treatment. Dermal tissues 30‐days after RF treatment exhibited prominent myofibroblast activity associated with collagen contractile activity during wound healing in addition to chronic inflammation. A decrease in the morphology of dermal collagen fibrils after RF treatment continued until seven days postoperatively. The collagen fibril diameter increased to near baseline at 30 days postoperatively. Low‐energy and multi‐pass treatments resulted in greater collagen fibril contraction and recovery at the nanostructural level at 30 days postoperatively than did a single high‐energy treatment. Microsc. Res. Tech. 76:219–224, 2013. © 2012 Wiley Periodicals, Inc.     

3D‐quantum interferometer using silicon microring‐embedded gold grating circuit

A 3D (three‐dimensional) quantum interferometer consisting of a silicon microring circuit proposed. The interferometer based on the electron spin cloud projections generated by microring‐embedded gold grating. The electron cloud oscillations result from the excitation of the gold grating at the center of the silicon microring by the dark soliton pulse of 1.50 μm center wavelength. The electron cloud spin‐down, spin‐up automatically formed in the two axes (x, y, respectively) and propagated along the z‐axis. In this proposal, the sensing mechanism of the circuit is manipulated by varying the reflector gold lengths of the sensing arm. The electron cloud spin coupled and changed by changing the gold lengths. The sensitivity measurement of the 3D quantum interferometer for three gold layer lengths of 100 nm, 500 nm, and 1,000 nm is (47.62 nm fs^?1, ±0.4762 fs^?1, ±0.01 nm^?1), (238.10 nm fs^?1, ±0.4762 fs^?1, ±0.002 nm^?1), (476.20 nm fs^?1, ±0.4762 fs^?1, ±0.001 nm^?1), respectively. The used circuit parameters are the real ones that can be fabricated by the currently available technology. Moreover, the silicon micro ring circuit acts as a plasmonic antenna, which can apply for wireless quantum communication. The electron cloud spin projection space–time control can apply for quantum cellular automata.     

Cornea and its adaptation to environment and accommodation function in veiled chameleon (Chamaeleo calyptratus): ultrastructure and 3D transmission electron tomography

We investigate the ultrastructural features and 3D electron tomography of chameleon (Chamaeleon calyptratus) which is a native of desert environments of Saudi Arabia. The corneas of the chameleon were fixed in 2.5% glutaraldehyde containing cuprolinic blue in sodium acetate buffer for electron microscopy and tomography, and observed under a JEOL 1400 transmission electron microscope. The thin cornea (21.92 μm) contained 28–30 collagen fibril lamellae. The middle stromal lamellae (from 13 to 19) contained keratocytes with a long cell process and filled with granular material. The CF diameter increased from lamella 1 (30.44 ± 1.03) to Lamella 5 (52.83 ± 2.00) then decreased towards the posterior stoma. The percentage of large CF diameters (55–65 nm) was very high in the lamellae L14 (38.8%) and L15 (85.7%). The mean PGs area of the posterior stroma (448.21 ± 24.84 nm²) was significantly larger than the mean PGs area of the anterior, (309.86 ± 8.2 nm²) and middle stroma 245.94 ± 8.28 nm²). 3D electron tomography showed the distribution of PGs around and over the CF. Variable diameters of CFs in the anterior stroma may provide compact lamellae which may restrict the low wavelength of light. Variable diameters of CFs in the anterior stroma may provide compact lamellae which may restrict the low wavelength of light. This accommodation function is achieved by bending of the cornea. During bending the anterior stroma was stretched and the posterior stroma was compressed due to the presence of small CFs. The middle stroma remained stiff due to the presence of large CFs. Large proteoglycans not only maintain hydration for a longer period of time, but also act as a lubricant to neutralise the shear forces in the anterior and posterior stroma during bending.     

TOC ‐ Issue Information

Knowledge of the collagen structure of an Achilles tendon is critical to comprehend the physiology, biomechanics, homeostasis and remodelling of the tissue. Despite intensive studies, there are still uncertainties regarding the microstructure. The majority of studies have examined the longitudinally arranged collagen fibrils as they are primarily attributed to the principal tensile strength of the tendon. Few studies have considered the structural integrity of the entire three‐dimensional (3D) collagen meshwork, and how the longitudinal collagen fibrils are integrated as a strong unit in a 3D domain to provide the tendons with the essential tensile properties. Using second harmonic generation imaging, a 3D imaging technique was developed and used to study the 3D collagen matrix in the midportion of Achilles tendons without tissue labelling and dehydration. Therefore, the 3D collagen structure is presented in a condition closely representative of the in vivo status. Atomic force microscopy studies have confirmed that second harmonic generation reveals the internal collagen matrix of tendons in 3D at a fibril level. Achilles tendons primarily contain longitudinal collagen fibrils that braid spatially into a dense rope‐like collagen meshwork and are encapsulated or wound tightly by the oblique collagen fibrils emanating from the epitenon region. The arrangement of the collagen fibrils provides the longitudinal fibrils with essential structural integrity and endows the tendon with the unique mechanical function for withstanding tensile stresses. A novel 3D microscopic method has been developed to examine the 3D collagen microstructure of tendons without tissue dehydrating and labelling. The study also provides new knowledge about the collagen microstructure in an Achilles tendon, which enables understanding of the function of the tissue. The knowledge may be important for applying surgical and tissue engineering techniques to tendon reconstruction.     

Particle size analysis: 90Y and 99mTc‐labelled colloids

Colloidal particle size is an important characteristic to consider when choosing a radiopharmaceutical for diagnosis and therapeutic purposes in nuclear medicine. Photon correlation spectroscopy (PCS) and transmission electron microscopy (TEM) were used to determine the particle‐size distribution of ⁹⁰Y‐ and ^99mTc‐labelled antimony trisulfide (Sb₂S₃) and tin colloids (Sn‐colloid). ⁹⁰Y‐Sb₂S₃ and ^99mTc‐Sb₂S₃ were found to have a diameter of 28.92 ± 0.14 and 35.61 ± 0.11 nm, respectively, by PCS. By TEM, ⁹⁰Y‐Sb₂S₃ particles were measured to be 14.33 ± 0.09 nm. ⁹⁰Y‐labelled Sn colloid were found to exist with a d_v(max1) of 805 nm and a d_v(max2) of 2590 nm, by PCS, whereas ^99mTc‐Sn colloid was shown to have more than 80% of radioactive particles of approximately 910 nm by PCS. For ⁹⁰Y‐labelled Sb₂S₃ and Sn colloid, a comparison of TEM and PCS indicates that these techniques found significantly different mean diameters. TEM has an excellent resolution necessary for radiocolloid particle‐sizing analysis, and it is a desirable size‐measuring technique because it is more reliable than PCS.     

The effect varied scanning electron microscopy desiccation techniques has on demineralized dentin

The study objective was to assess (a) the effect of a rubbing‐application of ethylenediaminetetraacetic acid (EDTA) or citric acid (CA) has on the ultrastructure of surface dentin and (b) the effect of two scanning electron microscopy (SEM) desiccation preparation techniques have on the collagen surface produced. Treatment regions on proximal root surfaces of extracted human teeth were root planned to expose dentin. Cotton pellets soaked in either 30% CA or 24% EDTA solution were rubbed on the treatment region then processed for SEM using one of two desiccation techniques, that is, (a) critically point dried from liquid CO₂ (control) or (b) air‐dried from tetramethylsilane (experimental). Specimens were coated with gold/palladium and viewed/photographed with an SEM. Specimens of the control groups displayed tufted fibrils (CA > EDTA) with many dentin tubules being partially obscured by overhanging fibrils. Air‐dried specimens of both treatment groups displayed a flat intact monolayer devoid of a matted meshwork of fibrous collagen. Discrete fibril “sprigs,” emanating from the surface monolayer, were characteristic of the EDTA group only. The rubbing‐application of EDTA on dentin produces a tufted fibril surface somewhat similar to that produced by CA. Air‐drying desiccation of both resulted in marked distortion with fibril collapse/coalescence of the tufted collagen matrix.     

Ultrastructural and three‐dimensional study of post‐LASIK ectasia cornea

INTRODUCTION: Post‐laser in situ keratomileusis (LASIK) corneal ectasia is a serious late postoperative complication. Here, we report the ultrastructural features of the post‐LASIK cornea of two patients. METHODS: Two normal corneas (age 24 and 37 years old) and two post‐LASIK ectaic corneas from two patients (A and B) were studied. The “patient A” (age 27 years) underwent penetrating keratoplasty and “patient B” (age 31 years) underwent deep‐anterior lamellar keratoplasty. The excised corneas were processed for light and electron microscopy. A total of 120 images for three‐dimensional (3D) reconstruction were taken by using the software “Recorder” and using a bottom mounted camera “Quemesa” attached to a JOEL 1400 transmission electron microscope. The 3D images were constructed using “Visual Kai” software. RESULTS: In the post‐LASIK cornea, the hemidesmosomes, the basement membrane, and Bowman”s layer were abnormal. The stromal lamellae were thin and disorganized. The collagen fibrils (CFs) diameter and interfibrillar spacing had decreased. Aggregated microfibrils were present in the Bowman's layer and all parts of the stroma. A large number of microfilaments were present at the detachment end of the flap and residual stroma. The 3D images showed the presence of collagen microfibrils and proteoglycans (PGs) within the CF of the normal and post‐LASIK cornea. The collagen microfibrils and PGs within the CFs had degenerated in the post‐LASIK cornea. CONCLUSION: Collagen microfibrils and PGs within the CFs were degenerated, leading to the degeneration of CFs, followed by the disorganization of lamellae in post‐LASIK cornea. The CFs diameter and interfibrillar spacing decreased. Microsc. Res. Tech. 77:91–98, 2014. © 2013 Wiley Periodicals, Inc.     

Observation of the ultrastructure of anterior cruciate ligament graft by atomic force microscopy

This study presented the fibril ultrastructure of retrieved grafts from the reconstruction of anterior cruciate ligament (ACL) using atomic force microscopy (AFM). The tapping mode images of the AFM were taken from different areas of the longitudinally cut grafts. Regular arrangement of collagen fibrils was found in certain areas of the graft. In many areas, however, the fibrils were not well arranged in a single direction, with some smaller fibrils oriented vertically to larger parallel fibrils. The crossing and tangling of fibrils in ACL grafts was well represented in the three‐dimensional AFM image. This abnormality of graft ultrastructure might indicate the possible alteration of the mechanical environment after ACL reconstruction. This study demonstrated the suitability and importance of ultrastructure observation of ACL grafts by AFM. SCANNING 31: 19–23, 2009. © 2009 Wiley Periodicals, Inc.     

Fluorescence relaxation in 3D from diffraction-limited sources of PAGFP or sinks of EGFP created by multiphoton photoconversion

The relaxation of fluorescence from diffraction‐limited sources of photoactivatable green fluorescent protein (PAGFP) or sinks of photobleached enhanced GFP (EGFP) created by multiphoton photo‐conversion was measured in solutions of varied viscosity (η), and in live, spherical Chinese hamster ovary (CHO) cells. Fluorescence relaxation was monitored with the probing laser fixed, or rapidly scanning along a line bisected by the photoconversion site. Novel solutions to several problems that hamper the study of PAGFP diffusion after multiphoton photoconversion are presented. A theoretical model of 3D diffusion in a sphere from a source in the shape of the measured multiphoton point‐spread function was applied to the fluorescence data to estimate the apparent diffusion coefficient, D_ap. The model incorporates two novel features that make it of broad utility. First, the model includes the no‐flux boundary condition imposed by cell plasma membranes, allowing assessment of potential impact of this boundary on estimates of D_ap. Second, the model uses an inhomogeneous source term that, for the first time, allows analysis of diffusion from sources produced by multiphoton photoconversion pulses of varying duration. For diffusion in aqueous solution, indistinguishable linear relationships between D_ap and η⁻¹ were obtained for the two proteins: for PAGFP, D_aq= 89 ± 2.4 μm² s⁻¹ (mean ± 95% confidence interval), and for EGFP D_aq= 91 ± 1.8 μm² s⁻¹. In CHO cells, the application of the model yielded D_ap= 20 ± 3 μm² s⁻¹ (PAGFP) and 19 ± 2 μm² s⁻¹ (EGFP). Furthermore, the model quantitatively predicted the decline in baseline fluorescence that accompanied repeated photobleaching cycles in CHO cells expressing EGFP, supporting the hypothesis of fluorophore depletion as an alternative to the oft invoked ‘bound fraction’ explanation of the deviation of the terminal fluorescence recovery from its pre‐bleach baseline level. Nonetheless for their identical diffusive properties, advantages of PAGFP over EGFP were found, including an intrinsically higher signal/noise ratio with 488‐nm excitation, and the requirement for ∼1/200th the cumulative light energy to produce data of comparable signal/noise.     

Real-time enzymatic degradation of human dentin collagen fibrils exposed to exogenous collagenase: an AFM study in situ

Objective: This study was carried out to observe the enzymatic degradation of human dentin collagen fibrils exposed to exogenous collagenase in situ using atomic force microscopy, to understand the characteristics of the enzymatic degradation of collagen fibrils on dentin specimens. Methods: Polished dentin specimens from caries‐free third molars were etched with citric acid, and then treated with an aqueous solution of 6.5% NaOCl for 120 s. The specimen was then put into a fluid cell and treated with a mixed solution of collagenase I (MMP‐1) and collagenase II (MMP‐8) for 9 h. AFM with contact mode was performed in situ to monitor the enzymatic degradation process of the dentin collagen fibrils. The distinctly topographic changes of the dentin surface were recorded continuously during different stages of the enzymatic degradation process. Results: The mixed solution of exogenous collagenase I and collagenase II could degrade dentin organic matrix (mainly collagen) efficiently, and the structures of dentin substrate were clearly exposed. Conclusion: It is possible to carry out real‐time observations on the enzymatic biodegradation process of human dentin collagen fibrils on dentin specimens with atomic force microscopy in situ. By this means, the fine structures of the etched dentin substrate were clearly revealed, possibly contributing to the related study of human dentin in vitro.     

Acute effects of splint immobilization of the forearm on in vivo microcirculation and histomorphology of the human skin

BACKGROUND: Splint immobilization of the forearm is often performed in clinical practice. Previous studies investigated the effect of immobilization on bone, cartilage, muscle, and tendon, however, the acute effects on human skin microcirculation and histomorphology remains elusive. METHODS: In 12 healthy, nonsmoking individuals (aged 29.7 ± 9.1 years) a randomly selected forearm was immobilized by splinting for 72 h, whereas the other forearm served as control. In vivo Reflectance‐Mode Confocal‐Microscopy (RMCM) was performed prior (baseline value) and postimmobilization to evaluate: quantitative blood cell flow; density of functional dermal capillaries; epidermal thickness; and granular cell size. RESULTS: At 72h forearm immobilization, quantitative blood cell flow was significantly reduced (42.86 ± 3.68 cells/min) compared to the control blood flow (53.11 ± 3.68 cells/min, P < 0.05) and dermal capillaries indicates less functional density (5.73 ± 0.63 capillaries/mm²) compared to the controls (7.04 ± 0.81 capillaries/mm², P < 0.05). Histometric assessment reveals significantly thinner epidermis following immobilization compared to the control site (40.02 ± 2.91 vs. 46.64±3.09 µm, P < 0.05). Granular cell size was significantly altered at 72 h splinting (730.1 ± 42.53 µm²) compared to the control cell size at 770.2 ± 38.21 µm². Comparison of baseline values of both forearms indicate statistically insignificance (P > 0.05) for each parameter. CONCLUSION: At 72 h splint immobilization, for the first time, significant adaptive mechanisms were evaluated on human skin microcirculation and histomorphology using in vivo RMCM. These adaptations may be considered as an incipient atrophy of the human skin. Long‐term effects of immobilization including the regenerative potential should be evaluated in further RMCM studies. Microsc. Res. Tech. 77:99–103, 2014. © 2013 Wiley Periodicals, Inc.     

Freeze-substitution of rabbit tibial articular cartilage reveals that radial zone collagen fibres are tubules

Investigations of the micromorphology of rabbit tibial articular cartilage using scanning and transmission electron microscopy revealed that the collagenous elements in the tissue form fluid-containing tubular structures. The commonly described radial or deep zone longitudinal fibres were found to be tubular structures with internal diameters of 1–2 μm. The walls of the tubules were composed of tightly packed fibrils of collagen. The tangential zone, close to the tibial plateau, was composed mainly of a spongy arrangement of collagen fibrils, containing bunches of tangentially lying small (< 1 μm) diameter tubules. The application of conventional chemical fixation techniques resulted in the fine detail of this tissue being obscured. When the tissue was frozen, followed by cryo-scanning electron microscopy or freeze-drying, prior to observation in the scanning electron microscope the tubule structures were not obviously present. It was only by applying freeze-substitution techniques, followed by critical point drying or resin embedding, that the structure was revealed clearly. Segregation of water into ice crystals did occur during the freezing process, but the formation of those crystals played no part in creating the tubular morphology observed. A similar structure was still revealed following pre-treatment with glycerol, methanol or Triton X-100, provided that concentration of these additives was not too high. The walls of the tubules in the radial region were composed of straight, longitudinally arranged as well as helically arranged, 30 nm diameter fibrils. The lumen of the tubules appears to be lined by a circumferentially arranged array of approximately 10 nm diameter fibres, spaced at regular intervals of 50–70 nm.     

Effects of Er:YAG laser on bond strength of self‐etching adhesives to caries‐affected dentin

The erbium:yttrium–aluminum–garnet (Er:YAG) laser may be effective the bond strength of adhesive systems on dentine surfaces, the chemical composition and aggressiveness of adhesive systems in clinical practice. The purpose of this study was to evaluate the effects of the Er:YAG laser system with the bonding ability of two different self‐etching adhesives to caries‐affected dentine in primary molars. Ninety mid‐coronal flat dentine surfaces obtained from sound and caries‐affected human primary dentine were treated with an Er:YAG laser or a bur. The prepared surfaces were restored with an adhesive system (Xeno V; Clearfil S³) and a compomer (Dyract Extra). The restored teeth were sectioned with a low‐speed saw and 162 samples were obtained. The bond strength of the adhesive systems was tested using the micro‐tensile test method. The data were statistically analyzed. A restored tooth in each group was processed for scanning electron microscopy evaluation. The values of the highest bond strength were obtained from the Clearfil S³‐Er:YAG laser‐sound dentine group in all groups. (24.57 ± 7.27 MPa) (P > 0.05). The values of the lowest bond strength were obtained from the Xeno V‐Er:YAG laser‐sound dentine group in all groups (11.01 ± 3.89 MPa). It was determined that the Clearfil S³ increased the bond strength on the surface applied with Er:YAG laser according to the Xeno V. Microsc. Res. Tech. 77:282–288, 2014. © 2014 Wiley Periodicals, Inc.     

Morphological,microscopic, and physicochemical studies of Diospyros montana

Adulteration is the root cause of producing not only a chemically and pharmacologically inferior but also in some instances hazardous or poisonous drug. Despite availability of several techniques, microscopy and physicochemical analyses are the most practical approaches for crude drug authentication. Hence, the present study aimed to evaluate morphological, microscopic, and physicochemical properties of root, bark, leaf, and fruit of Diospyros montana Morphological properties were determined by sensory organs, whereas microscopic features of cross‐sections and powders were determined by light and scanning electron microscopy. The proximate and fluorescence analyses were performed using the standard guidelines. The physical examination of fresh, shade‐dried, and powdered material showed no significant change in color. The identifying cellular structures included cuboidal cork, pitted tracheids, scalariform, reticulate and spiral xylary vessels, and rosettes, raphide, and cuboidal calcium oxalate crystals. The stomatal number, stomatal index, vein‐islet and vein‐termination number, and palisade ratio in the leaf were found to be 293.91 ± 32.68 mm^?2, 64.18 ± 3.42%, 22.00 ± 3.81 mm^?2 and 38.40 ± 5.81 mm^?2, and 3.85 ± 0.60, respectively. Total ash, acid insoluble ash, water soluble ash and sulfated ash of leaf (9.00 ± 0.50%, 1.67 ± 0.23%, 2.00 ± 0.22% and 14.50 ± 0.99%, respectively), foaming index of bark and root (111.11 ± 2.11), and swelling index of fruit (19.00 ± 3.45) were higher than the other parts. The powder of different parts showed characteristic colors in the daylight and UV light upon treatment with various regents. The plant was found to be rich in saponins, fibers, and flavonoids. The results of the present study may serve as identifiers of different parts of Diospyros montana.