Evaluation of the Morphological Characteristics of Laser-Irradiated Dentin

OBJECTIVE The aim of this study was to investigate the effect of different energy settings of Er:YAG laser irradiation on dentin surface morphology with respect to the number of opened dentinal tubules. BACKGROUND DATA An ideally prepared dentin surface with opened dentinal tubules is a prerequisite for adhesive fixation. No study, however, has yet compared the numbers of opened dentinal tubules with regard to statistical differences. METHODS Conventional preparations using a bur with or without additional acid etching acted as control groups. Dentin specimens were prepared from human third molars and randomly divided into eight groups according to the energy settings of the laser (1, 1.5, 4, 6, 7.5, and 8 W) and two controls (bur and bur plus acid etching). After surface preparation, dentin surfaces were analyzed with a scanning electron microscope, and the number of opened dentinal tubules in a defined area was counted. RESULTS The control groups showed smooth surfaces with (bur plus acid etching) and without opened dentinal tubules (bur), whereas all laser-irradiated surfaces showed rough surfaces. Using the energy setting of 4 W resulted in significantly more opened dentinal tubules than the conventional preparation technique using the bur with additional acid etching. In contrast, the energy setting of 8 W showed significantly fewer opened dentinal tubules, and also exhibited signs of thermal damage. CONCLUSIONS The Er:YAG laser with an energy setting of 4 W generates a dentin surface with opened dentinal tubules, a prerequisite for adhesive fixation.

RacF1, a Novel Member of the Rho Protein Family in Dictyostelium discoideum, Associates Transiently with Cell Contact Areas, Macropinosomes, and Phagosomes

Using a PCR approach we have isolated racF1, a novel member of the Rho family in Dictyostelium. The racF1 gene encodes a protein of 193 amino acids and is constitutively expressed throughout the Dictyostelium life cycle. Highest identity (94%) was found to a RacF2 isoform, to Dictyostelium Rac1A, Rac1B, and Rac1C (70%), and to Rac proteins of animal species (64–69%). To investigate the role of RacF1 in cytoskeleton-dependent processes, we have fused it at its amino-terminus with green fluorescent protein (GFP) and studied the dynamics of subcellular redistribution using a confocal laser scanning microscope and a double-view microscope system. GFP–RacF1 was homogeneously distributed in the cytosol and accumulated at the plasma membrane, especially at regions of transient intercellular contacts. GFP–RacF1 also localized transiently to macropinosomes and phagocytic cups and was gradually released within <1 min after formation of the endocytic vesicle or the phagosome, respectively. On stimulation with cAMP, no enrichment of GFP–RacF1 was observed in leading fronts, from which it was found to be initially excluded. Cell lines were obtained using homologous recombination that expressed a truncated racF1 gene lacking sequences encoding the carboxyl-terminal region responsible for membrane targeting. These cells displayed normal phagocytosis, endocytosis, and exocytosis rates. Our results suggest that RacF1 associates with dynamic structures that are formed during pinocytosis and phagocytosis. Although RacF1 appears not to be essential, it might act in concert and/or share functions with other members of the Rho family in the regulation of a subset of cytoskeletal rearrangements that are required for these processes.

Localization and movement of mineral oil in plants by fluorescence and confocal microscopy

Fluorescence and confocal laser scanning microscopy were explored to investigate the movement and localization of mineral oils in citrus. In a laboratory experiment, fluorescence microscopy observation indicated that when a 'narrow' distillation fraction of an nC23 horticultural mineral oil was applied to adaxial and opposing abaxial leaf surfaces of potted orange [Citrus x aurantium L. (Sapindales: Rutaceae)] trees, oil penetrated steadily into treated leaves and, subsequently, moved to untreated petioles of the leaves and adjacent untreated stems. In another experiment, confocal laser scanning microscopy was used to visualize the penetration into, and the subsequent cellular distribution of, an nC24 agricultural mineral oil in C. trifoliata L. seedlings. Oil droplets penetrated or diffused into plants via both stomata and the cuticle of leaves and stems, and then moved within intercellular spaces and into various cells including phloem and xylem. Oil accumulated in droplets in intercellular spaces and within cells near the cell membrane. Oil entered cells without visibly damaging membranes or causing cell death. In a field experiment with mature orange trees, droplets of an nC23 horticultural mineral oil were observed, by fluorescence microscopy, in phloem sieve elements in spring flush growth produced 4-5 months and 16-17 months after the trees were sprayed with oil. These results suggest that movement of mineral oil in plants is both apoplastic via intercellular spaces and symplastic via plasmodesmata. The putative pattern of the translocation of mineral oil in plants and its relevance to oil-induced chronic phytotoxicity are discussed.

Femtosecond laser-induced microstructures on diamond for microfluidic sensing devices applications

This paper reported a three-dimensional microfluidic channel structure, which was fabricated by Yb:YAG 1026?nm femtosecond laser irradiation on a single-crystalline diamond substrate. The femtosecond laser irradiation energy level was optimized at 100?kHz repetition rate with a sub-500 femtosecond pulse duration. The morphology and topography of the microfluidic channel were characterized by a scanning electron microscope and an atomic force microscope. Raman spectroscopy indicated that the irradiated area was covered by graphitic materials. By comparing the cross-sectional profiles before/after removing the graphitic materials, it could be deduced that the microfluidic channel has an average depth of ~410?nm with periodical ripples perpendicular to the irradiation direction. This work proves the feasibility of using ultra-fast laser inscription technology to fabricate microfluidic channels on biocompatible diamond substrates, which offers a great potential for biomedical sensing applications.

Femtosecond laser-induced microstructures on diamond for microfluidic sensing device applications

This paper reported a three-dimensional microfluidic channel structure, which was fabricated by Yb:YAG 1026?nm femtosecond laser irradiation on a single-crystalline diamond substrate. The femtosecond laser irradiation energy level was optimized at 100?kHz repetition rate with a sub-500 femtosecond pulse duration. The morphology and topography of the microfluidic channel were characterized by a scanning electron microscope and an atomic force microscope. Raman spectroscopy indicated that the irradiated area was covered by graphitic materials. By comparing the cross-sectional profiles before/after removing the graphitic materials, it could be deduced that the microfluidic channel has an average depth of ~410?nm with periodical ripples perpendicular to the irradiation direction. This work proves the feasibility of using ultra-fast laser inscription technology to fabricate microfluidic channels on biocompatible diamond substrates, which offers a great potential for biomedical sensing applications.

Femtosecond laser-induced microstructures on diamond for microfluidic sensing devices applications

This paper reported a three-dimensional microfluidic channel structure, which was fabricated by Yb:YAG 1026?nm femtosecond laser irradiation on a single-crystalline diamond substrate. The femtosecond laser irradiation energy level was optimized at 100?kHz repetition rate with a sub-500 femtosecond pulse duration. The morphology and topography of the microfluidic channel were characterized by a scanning electron microscope and an atomic force microscope. Raman spectroscopy indicated that the irradiated area was covered by graphitic materials. By comparing the cross-sectional profiles before/after removing the graphitic materials, it could be deduced that the microfluidic channel has an average depth of ~410?nm with periodical ripples perpendicular to the irradiation direction. This work proves the feasibility of using ultra-fast laser inscription technology to fabricate microfluidic channels on biocompatible diamond substrates, which offers a great potential for biomedical sensing applications.

In Situ Observation of the Thermal Decomposition of Weddelite by Heating Stage Environmental Scanning Electron Microscopy

The morphological and chemical changes occurring during the thermal decomposition of weddelite, CaC2O4·2H2O, have been followed in real time in a heating stage attached to an Environmental Scanning Electron Microscope operating at a pressure of 2 Torr, with a heating rate of 10 °C/min and an equilibration time of approximately 10 min. The dehydration step around 120 °C and the loss of CO around 425 °C do not involve changes in morphology, but changes in the composition were observed. The final reaction of CaCO3 to CaO while evolving CO2 around 600 °C involved the formation of chains of very small oxide particles pseudomorphic to the original oxalate crystals. The change in chemical composition could only be observed after cooling the sample to 350 °C because of the effects of thermal radiation.

The osteogenic properties of CaP/silk composite scaffolds

The rationale for the present study was to develop porous CaP/silk composite scaffolds with a CaP-phase distribution and pore architecture better suited to facilitate osteogenic properties of human bone mesenchymal stromal cells (BMSCs) and in vivo bone formation abilities. This was achieved by first preparing CaP/silk hybrid powders which were then incorporated into silk to obtain uniform CaP/silk composite scaffolds, by means of a freeze-drying method. The composition, microstructure and mechanical properties of the CaP/silk composite scaffolds were ascertained by X-ray diffraction (XRD), Fourier transform infrared spectra (FTIR), scanning electron microscope (SEM) and a universal mechanical testing machine. BMSCs were cultured in these scaffolds and cell proliferation analyzed by confocal microscopy and MTS assay. Alkaline phosphatase (ALP) activity and osteogenic gene expression were assayed to determine if osteogenic differentiation had taken place. A calvarial defect model in SCID mice was used to determine the in vivo bone forming ability of the hybrid CaP/silk scaffolds. Our results showed that incorporating the hybrid CaP/silk powders into silk scaffolds improved both pore structure architecture and distribution of CaP powders in the composite scaffolds. By incorporating the CaP phase into silk scaffolds in vitro osteogenic differentiation of BMSCs was enhanced and there was increased in vivo cancellous bone formation. Here we report a method with which to prepare Ca/P composite scaffolds with a pore structure and Ca/P distribution better suited to facilitate BMSC differentiation and bone formation.

Scanning electron microscopic study of microstructure of Gala apples during hot air drying

Microscopic changes occur in plant food materials during drying significantly influence the macroscopic properties and quality factors of the dried food materials. It is very critical to study microstructure to understand the underlying cellular mechanisms to improve performance of the food drying techniques. However, there is very limited research conducted on such microstructural changes of plant food material during drying. In this work, Gala apple parenchyma tissue samples were studied using a scanning electron microscope for gradual microstructural changes as affected by temperature, time and moisture content during hot air drying at two drying temperatures: 57 ℃ and 70 ℃. For fresh samples, the average cellular parameter values were; cell area: 20000 μm2, ferret diameter: 160 μm, perimeter: 600 μm, roundness: 0.76, elongation: 1.45 and compactness: 0.84. During drying, a higher degree of cell shrinkage was observed with cell wall warping and increase in intercellular space. However, no significant cell wall breakage was observed. The overall reduction of cell area, ferret diameter and perimeter were about 60%, 40% and 30%. The cell roundness and elongation showed overall increments of about 5% and the compactness remained unchanged. Throughout the drying cycle, cellular deformations were mainly influenced by the moisture content. During the initial and intermediate stages of drying, cellular deformations were also positively influenced by the drying temperature and the effect was reversed at the final stages of drying which provides clues for case hardening of the material.

Stress effects caused by the expression of a mutant cellobiohydrolase I and proteasome inhibition in Trichoderma reesei Rut-C30

Trichoderma reesei Rut-C30 is used widely as an expression host for various gene products. We have explored cellular effects caused by the expression of a mutant form of cellobiohydrolase I (CBHI), the major secreted protein of T. reesei using biochemical and transcriptomic analyses and confocal laser scanning microscopy. The mutated CBHI was tagged fluorescently with Venus to establish the subcellular location of the fusion protein and its potential association with the proteasome, an organelle assigned for the disposal of misfolded proteins. Expression of the mutant CBHI in the high protein-secreting host Rut-C30 caused physiological changes in the fungal hyphae, affected protein secretion and elicited ER stress. A massive upregulation of UPR- and ERAD-related genes sec61, der1, uba1, bip1, pdi1, prp1, cxl1 and lhs1 was observed by qRT-PCR in the CBHIΔ4-Venus strain with four mutations introduced in the DNA encoding the core domain of CBHI. Further stress was applied to this strain by inhibiting function of the proteasome with MG132 (N-benzoylcarbonyl(Cbz)-Leu-Leu-leucinal). The effect of MG132 was found to be specific to the proteasome-associated genes. There are no earlier reports on the effect of proteasome inhibition on protein quality control in filamentous fungi. Confocal fluorescence microscopy studies suggested that the mutant CBHI accumulated in the ER and colocalized with the fungal proteasome. These results provide an indication that there is a limit to how far T. reesei Rut-C30, already under secretion stress, can be pressed to produce higher protein yields.

Non-invasive techniques for imaging in-vivo human corneal sub basal nerve mapping and migration rate in diabetic neuropathy

Purpose Corneal confocal microscopy (CCM) is a rapid non-invasive ophthalmic technique, which has been shown to diagnose and stratify the severity of diabetic neuropathy. Current morphometric techniques assess individual static images of the subbasal nerve plexus; this work explores the potential for non-invasive assessment of the wide-field morphology and dynamic changes of this plexus in vivo. Methods In this pilot study, laser scanning CCM was used to acquire maps (using a dynamic fixation target and semi-automated tiling software) of the central corneal sub-basal nerve plexus in 4 diabetic patients with and 6 without neuropathy and in 2 control subjects. Nerve migration was measured in an additional 7 diabetic patients with neuropathy, 4 without neuropathy and in 2 control subjects by repeating a modified version of the mapping procedure within 2-8 weeks, thus facilitating re-identification of distinctive nerve landmarks in the 2 montages. The rate of nerve movement was determined from these data and normalised to a weekly rate (µm/week), using customised software. Results Wide-field corneal nerve fibre length correlated significantly with the Neuropathy Disability Score (r = -0.58, p < 0.05), vibration perception (r = -0.66, p < 0.05) and peroneal conduction velocity (r = 0.67, p < 0.05). Central corneal nerve fibre length did not correlate with any of these measures of neuropathy (p > 0.05 for all). The rate of corneal nerve migration was 14.3 ± 1.1 µm/week in diabetic patients with neuropathy, 19.7 ± 13.3µm/week in diabetic patients without neuropathy, and 24.4 ± 9.8µm/week in control subjects; however, these differences were not significantly different (p = 0.543). Conclusions Our data demonstrate that it is possible to capture wide-field images of the corneal nerve plexus, and to quantify the rate of corneal nerve migration by repeating this procedure over a number of weeks. Further studies on larger sample sizes are required to determine the utility of this approach for the diagnosis and monitoring of diabetic neuropathy.

Efficiently capturing large, complex cultural heritage sites with a handheld mobile 3D laser mapping system

Accurate three-dimensional representations of cultural heritage sites are highly valuable for scientific study, conservation, and educational purposes. In addition to their use for archival purposes, 3D models enable efficient and precise measurement of relevant natural and architectural features. Many cultural heritage sites are large and complex, consisting of multiple structures spatially distributed over tens of thousands of square metres. The process of effectively digitising such geometrically complex locations requires measurements to be acquired from a variety of viewpoints. While several technologies exist for capturing the 3D structure of objects and environments, none are ideally suited to complex, large-scale sites, mainly due to their limited coverage or acquisition efficiency. We explore the use of a recently developed handheld mobile mapping system called Zebedee in cultural heritage applications. The Zebedee system is capable of efficiently mapping an environment in three dimensions by continually acquiring data as an operator holding the device traverses through the site. The system was deployed at the former Peel Island Lazaret, a culturally significant site in Queensland, Australia, consisting of dozens of buildings of various sizes spread across an area of approximately 400 × 250 m. With the Zebedee system, the site was scanned in half a day, and a detailed 3D point cloud model (with over 520 million points) was generated from the 3.6 hours of acquired data in 2.6 hours. We present results demonstrating that Zebedee was able to accurately capture both site context and building detail comparable in accuracy to manual measurement techniques, and at a greatly increased level of efficiency and scope. The scan allowed us to record derelict buildings that previously could not be measured because of the scale and complexity of the site. The resulting 3D model captures both interior and exterior features of buildings, including structure, materials, and the contents of rooms.

Fully automated, semiautomated, and manual morphometric analysis of corneal subbasal nerve plexus in individuals with and without diabetes

Purpose The aim of the study was to determine the association, agreement, and detection capability of manual, semiautomated, and fully automated methods of corneal nerve fiber length (CNFL) quantification of the human corneal subbasal nerve plexus (SNP). Methods Thirty-three participants with diabetes and 17 healthy controls underwent laser scanning corneal confocal microscopy. Eight central images of the SNP were selected for each participant and analyzed using manual (CCMetrics), semiautomated (NeuronJ), and fully automated (ACCMetrics) software to quantify the CNFL. Results For the entire cohort, mean CNFL values quantified by CCMetrics, NeuronJ, and ACCMetrics were 17.4 ± 4.3 mm/mm2, 16.0 ± 3.9 mm/mm2, and 16.5 ± 3.6 mm/mm2, respectively (P < 0.01). CNFL quantified using CCMetrics was significantly higher than those obtained by NeuronJ and ACCMetrics (P < 0.05). The 3 methods were highly correlated (correlation coefficients 0.87–0.98, P < 0.01). The intraclass correlation coefficients were 0.87 for ACCMetrics versus NeuronJ and 0.86 for ACCMetrics versus CCMetrics. Bland–Altman plots showed good agreement between the manual, semiautomated, and fully automated analyses of CNFL. A small underestimation of CNFL was observed using ACCMetrics with increasing the amount of nerve tissue. All 3 methods were able to detect CNFL depletion in diabetic participants (P < 0.05) and in those with peripheral neuropathy as defined by the Toronto criteria, compared with healthy controls (P < 0.05). Conclusions Automated quantification of CNFL provides comparable neuropathy detection ability to manual and semiautomated methods. Because of its speed, objectivity, and consistency, fully automated analysis of CNFL might be advantageous in studies of diabetic neuropathy.

X-ray diffraction of permalloy nanoparticles fabricated by laser ablation in water

Permalloy (NiFeMo) nanoparticles were fabricated by laser ablation of bulk material in water with a UV pulsed laser. Transmission electron microscope images showed that approximately spherical particles about 50 nm in diameter were formed in the ablation process. All diffraction peaks corresponding to the bulk material were present in the nanoparticles. In addition to these peaks several new peaks were observed in the nanoparticles, which were attributed to nickel oxide.

Normative values for corneal nerve morphology assessed using corneal confocal microscopy: A multinational normative data set

OBJECTIVE Corneal confocal microscopy is a novel diagnostic technique for the detection of nerve damage and repair in a range of peripheral neuropathies, in particular diabetic neuropathy. Normative reference values are required to enable clinical translation and wider use of this technique. We have therefore undertaken a multicenter collaboration to provide worldwide age-adjusted normative values of corneal nerve fiber parameters. RESEARCH DESIGN AND METHODS A total of 1,965 corneal nerve images from 343 healthy volunteers were pooled from six clinical academic centers. All subjects underwent examination with the Heidelberg Retina Tomograph corneal confocal microscope. Images of the central corneal subbasal nerve plexus were acquired by each center using a standard protocol and analyzed by three trained examiners using manual tracing and semiautomated software (CCMetrics). Age trends were established using simple linear regression, and normative corneal nerve fiber density (CNFD), corneal nerve fiber branch density (CNBD), corneal nerve fiber length (CNFL), and corneal nerve fiber tortuosity (CNFT) reference values were calculated using quantile regression analysis. RESULTS There was a significant linear age-dependent decrease in CNFD (-0.164 no./mm(2) per year for men, P < 0.01, and -0.161 no./mm(2) per year for women, P < 0.01). There was no change with age in CNBD (0.192 no./mm(2) per year for men, P = 0.26, and -0.050 no./mm(2) per year for women, P = 0.78). CNFL decreased in men (-0.045 mm/mm(2) per year, P = 0.07) and women (-0.060 mm/mm(2) per year, P = 0.02). CNFT increased with age in men (0.044 per year, P < 0.01) and women (0.046 per year, P < 0.01). Height, weight, and BMI did not influence the 5th percentile normative values for any corneal nerve parameter. CONCLUSIONS This study provides robust worldwide normative reference values for corneal nerve parameters to be used in research and clinical practice in the study of diabetic and other peripheral neuropathies.