Effect of short training on vaginal fluid microscopy (wet mount) learning

Objective: Is it feasible to learn the basics of wet mount microscopy of vaginal fluid in 10 hours?Materials and Methods: This is a pilot project wherein 6 students with different grades of education were invited for being tested on their ability to read wet mount microscopic slides before and after 10 hours of hands-on training. Microscopy was performed according to a standard protocol (Femicare, Tienen, Belgium). Before and after training, all students had to evaluate a different set of 50 digital slides. Different diagnoses and microscopic patterns had to be scored. kappa indices were calculated compared with the expert reading. Results: All readers improved their mean scores significantly, especially for the most important types of altered flora (p < .0001). The mean increase in reading concordance (kappa from 0.64 to 0.75) of 1 student with a solid previous experience with microscopy did not reach statistical significance, but the remaining 5 students all improved their scores from poor performance (all kappa < 0.20) to moderate (kappa = 0.53, n = 1) to good (kappa > 0.61, n = 4) concordance. Reading quality improved and reached fair to good concordance on all microscopic items studied, except for the detection of parabasal cells and cytolytic flora. Conclusions: Although further improvement is still possible, a short training course of 10 hours enables vast improvement on wet mount microscopy accuracy and results in fair to good concordance of the most important variables of the vaginal flora compared to a reference reader.

Evaluation of early changes induced by diuron in the rat urinary bladder using different processing methods for scanning electron microscopy

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Alveolar bone dynamics in osteoporotic rats treated with raloxifene or alendronate: confocal microscopy analysis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Early development of Betta splendens under stereomicroscopy and scanning electron microscopy

Betta splendens is a very important ornamental species. The current paper describes the embryonic and larval development of B. splendens under stereomicroscopy and scanning electron microscopy. Eggs and larvae from natural spawning were collected at different developmental stages at previously established intervals and analysed. The eggs of B. splendens are yellowish, clear, spherical, demersal, translucent and telolecithal with a large amount of yolk. Between 0-2 h post-initial collection (hpIC), the eggs were at the egg cell, first cleavage and morula stages. The blastula stage was identified at 2-3 hpIC and the early gastrula phase was observed at 3-4 hpIC with 20% epiboly, which was finalized after 13-18 hpIC. When the pre-larvae were ready to hatch, the appearance of somites and the free tail were observed, at 23-25 hpIC. At 29 hpIC, the majority of larvae had already hatched at an average temperature of 28.4 +/- 0.2 degrees C. The newly hatched larvae measured 2.47 +/- 0.044 mm total length. The mouth opened at 23 h post-hatching (hPH) and the yolk sac was totally absorbed at 73 hPH. After 156 hPH, the heart was pumping blood throughout the entire larval body. The caudal fin, operculum and eyes were well developed at 264 hPH. When metamorphosis was complete at 768 hPH, the larvae became juveniles. The current study presents the first results about early development of B. splendens and provides relevant information for its reproduction, rearing and biology.

Digital Inline Holographic Microscopy (DIHM) of weakly-scattering subjects

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Evaluation of the bleached human enamel by scanning electron microscopy

Since bleaching has become a popular procedure, the effect of peroxides on dental hard tissues is of great interest in research. Purpose: The aim of this in vitro study was to perform a qualitative analysis of the human enamel after the application of in-office bleaching agents, using Scanning Electron Microscopy (SEM). Materials and Methods: Twenty intact human third molars extracted for orthodontic reasons were randomly divided into four groups (n=5) treated as follows: G1- storage in artificial saliva (control group); G2- four 30-minute applications of 35% carbamide peroxide (total exposure: 2h); G3- four 2-hour exposures to 35% carbamide peroxide (total exposure: 8h); G4- two applications of 35% hydrogen peroxide, which was light-activated with halogen lamp at 700mW/cm2 during 7min and remained in contact with enamel for 20min (total exposure: 40min). All bleaching treatments adopted in this study followed the application protocols advised by manufacturers. Evaluation of groups submitted to 35% carbamide peroxide was carried out after two time intervals (30 minutes and 2 hours per session), following the extreme situations recommended by the manufacturer. Specimens were prepared for SEM analysis performing gold sputter coating under vacuum and were examined using 15kV at 500x and 2000x magnification. Results: Morphological alterations on the enamel surface were similarly detected after bleaching with either 35% carbamide peroxide or 35% hydrogen peroxide. Surface porosities were characteristic of an erosive process that took place on human enamel. Depression areas, including the formation of craters, and exposure of enamel rods could also be detected. Conclusion: Bleaching effects on enamel morphology were randomly distributed throughout enamel surface and various degrees of enamel damage could be noticed. Clinical significance: In-office bleaching materials may adversely affect enamel morphology and therefore should be used with caution.

Bone regeneration in surgically created deffects filled with autogenous bone: epifluorescence microscopy analysis in rats

Although the search for the ideal bone substitute has been the focus of a large number of studies, autogenous bone is still the gold standard for the filling of defects caused by pathologies and traumas, and mainly, for alveolar ridge reconstruction, allowing the titanium implants installation. OBJECTIVES: The aim of this study was to evaluate the dynamics of autogenous bone graft incorporation process to surgically created defects in rat calvaria, using epifluorescence microscopy. MATERIAL AND METHODS: Five adult male rats weighing 200-300 g were used. The animals received two 5-mm-diameter bone defects bilaterally in each parietal bone with a trephine bur under general anesthesia. Two groups of defects were formed: a control group (n=5), in which the defects were filled with blood clot, and a graft group (n=5), in which the defects were filled with autogenous bone block, removed from the contralateral defect. The fluorochromes calcein and alizarin were applied at the 7th and 30th postoperative days, respectively. The animals were killed at 35 days. RESULTS: The mineralization process was more intense in the graft group (32.09%) and occurred mainly between 7 and 30 days, the period labeled by calcein (24.66%). CONCLUSIONS: The fluorochromes showed to be appropriate to label mineralization areas. The interfacial areas between fluorochrome labels are important sources of information about the bone regeneration dynamics.

Confocal laser scanning microscopy investigation of bond interfaces involved in fiber glass post cementation

Objective: This confocal microscopy study evaluated the cement/dentin and cement/post interfaces along theroot canalwallswhenfiberglasspostswerebonded to dentin using different types of cements. Material & Methods: Thirty endodontically treated premolars were divided into 3 groups according to the adhesive materials used in the bonding procedure: Prime & Bond 2.1/Self Cure + Enforce, RelyX Unicem and RelyX Luting. Rhodamine B dye was incorporated in the luting materials for the cementation of the fiber glass posts (Exacto, Angelus) to dentin. Three transversal slices (apical, middle and coronal) were examined under confocal laser scanning microscopy. Statistical analysis was performed using the Kappa, Kruskal-Wallis and Dunnet tests, in a significance level of 5%. Results: The Prime & Bond 2.1/Self Cure + Enforce presented a uniform formation of tags in the dentin but gaps in the cement/dentin interface. The RelyX Unicem and RelyX Luting presented an adhesive interface with a fewer amount of gaps, but showed shorter tag formation than the Enforce system. All cements presented the same pattern of bubbles inside the cements. The RelyX Luting presented a greater amount of cracks inside the cement in comparison with the other cements in the coronal third, while no difference was observed between RelyX Unicem and Enforce. The RelyX Luting showed the lowest quantity of cement penetration into the post. Conclusion: In general, the quality of bonding interfaces of fiber posts luted to root canals was affected by both location and type of cement.

Image-Guided Cryoablation of the Spine in a Swine Model: Clinical, Radiological, and Pathological Findings with Light and Electron Microscopy

This study was designed to present the feasibility of an in vivo image-guided percutaneous cryoablation of the porcine vertebral body. Methods The institutional animal care committee approved this study. Cone-beam computed tomography (CBCT)-guided vertebral cryoablations (n = 22) were performed in eight pigs with short, 2-min, single or double-freezing protocols. Protective measures to nerves included dioxide carbon (CO2) epidural injections and spinal canal temperature monitoring. Clinical, radiological, and pathological data with light (n = 20) or transmission electron (n = 2) microscopic analyses were evaluated after 6 days of clinical follow-up and euthanasia. Results CBCT/fluoroscopic-guided transpedicular vertebral body cryoprobe positioning and CO2 epidural injection were successful in all procedures. No major complications were observed in seven animals (87.5 %, n = 8). A minor complication was observed in one pig (12.5 %, n = 1). Logistic regression model analysis showed the cryoprobe-spinal canal (Cp-Sc) distance as the most efficient parameter to categorize spinal canal temperatures lower than 19 °C (p<0.004), with a significant Pearson’s correlation test (p < 0.041) between the Cp-Sc distance and the lowest spinal canal temperatures. Ablation zones encompassed pedicles and the posterior wall of the vertebral bodies with an inflammatory rim, although no inflammatory infiltrate was depicted in the surrounding neural structures at light microscopy. Ultrastructural analyses evidenced myelin sheath disruption in some large nerve fibers, although neurological deficits were not observed. Conclusions CBCT-guided vertebral cryoablation of the porcine spine is feasible under a combination of a short freezing protocol and protective measures to the surrounding nerves. Ultrastructural analyses may be helpful assess the early modifications of the nerve fibers.

Environmental effects in kelvin force microscopy of modified diamond surfaces

We have explored the effects of atmospheric environment on Kelvin force microscopy (KFM) measurements of potential difference between different regions of test polycrystalline diamond surfaces. The diamond films were deposited by microwave plasma-assisted chemical vapor deposition, which naturally produces hydrogen terminations on the surface of the films formed. Selected regions were patterned by electron-beam lithography and chemical terminations of oxygen or fluorine were created by exposure to an oxygen or fluorine plasma source. For KFM imaging, the samples were mounted in a hood with a constant flow of helium gas. Successive images were taken over a 5-h period showing the effect of the environment on KFM imaging. We conclude that the helium flow removes water molecules adsorbed on the surface of the samples, resulting in differences in surface potential between adjacent regions. The degree of water removal is different for surfaces with different terminations. The results highlight the importance of taking into account the atmospheric environment when carrying out KFM analysis. (C) 2012 Wiley Periodicals, Inc.

Liquid Crystal Nanodispersions Enable the Cutaneous Delivery of Photosensitizer for Topical PDT: Fluorescence Microscopy Study of Skin Penetration

Topical photodynamic therapy (PDT) has been applied to almost all types of nonmelanoma skin cancer and numerous superficial benign skin disorders. Strategies to improve the accumulation of photosensitizer in the skin have been studied in recent years. Although the hydrophilic phthalocyanine zinc compound, zinc phthalocyanine tetrasulfonate (ZnPcSO4) has shown high photodynamic efficiency and reduced phototoxic side effects in the treatment of brain tumors and eye conditions, its use in topical skin treatment is currently limited by its poor skin penetration. In this study, nanodispersions of monoolein (MO)-based liquid crystalline phases were studied for their ability to increase ZnPcSO4 uptake by the skin. Lamellar, hexagonal and cubic crystalline phases were prepared and identified by polarizing light microscopy, and the nanodispersions were analyzed by dynamic light scattering. In vitro skin penetration studies were performed using a Franz's cell apparatus, and the skin uptake was evaluated in vivo in hairless mice. Aqueous dispersions of cubic and hexagonal phases showed particles of nanometer size, approximately 224 +/- 10 nm and 188 +/- 10 nm, respectively. In vitro skin retention experiments revealed higher fluorescence from the ZnPcSO4 in deeper skin layers when this photosensitizer was loaded in the hexagonal nanodispersion system when compared to both the cubic phase nanoparticles and the bulk crystalline phases (lamellar, cubic and hexagonal). The hexagonal nanodispersion showed a similar penetration behavior in animal tests. These results are important findings, suggesting the development of MO liquid crystal nanodispersions as potential delivery systems to enhance the efficacy of topical PDT.

The Endothelial Sample Size Analysis in Corneal Specular Microscopy Clinical Examinations

Purpose: To evaluate endothelial cell sample size and statistical error in corneal specular microscopy (CSM) examinations. Methods: One hundred twenty examinations were conducted with 4 types of corneal specular microscopes: 30 with each BioOptics, CSO, Konan, and Topcon corneal specular microscopes. All endothelial image data were analyzed by respective instrument software and also by the Cells Analyzer software with a method developed in our lab(US Patent). A reliability degree (RD) of 95% and a relative error (RE) of 0.05 were used as cut-off values to analyze images of the counted endothelial cells called samples. The sample size mean was the number of cells evaluated on the images obtained with each device. Only examinations with RE<0.05 were considered statistically correct and suitable for comparisons with future examinations. The Cells Analyzer software was used to calculate the RE and customized sample size for all examinations. Results: Bio-Optics: sample size, 97 +/- 22 cells; RE, 6.52 +/- 0.86; only 10% of the examinations had sufficient endothelial cell quantity (RE<0.05); customized sample size, 162 +/- 34 cells. CSO: sample size, 110 +/- 20 cells; RE, 5.98 +/- 0.98; only 16.6% of the examinations had sufficient endothelial cell quantity (RE<0.05); customized sample size, 157 +/- 45 cells. Konan: sample size, 80 +/- 27 cells; RE, 10.6 +/- 3.67; none of the examinations had sufficient endothelial cell quantity (RE>0.05); customized sample size, 336 +/- 131 cells. Topcon: sample size, 87 +/- 17 cells; RE, 10.1 +/- 2.52; none of the examinations had sufficient endothelial cell quantity (RE>0.05); customized sample size, 382 +/- 159 cells. Conclusions: A very high number of CSM examinations had sample errors based on Cells Analyzer software. The endothelial sample size (examinations) needs to include more cells to be reliable and reproducible. The Cells Analyzer tutorial routine will be useful for CSM examination reliability and reproducibility.

Probing the Enamel Topography After Acid Erosion by Scanning Electrochemical Microscopy

In this work, we present an investigation on the thickness of the eroded enamel layer in tooth samples after exposure to citric and hydrochloric acid by using Scanning Electrochemical Microscopy (SECM). Approaching curves with typical negative feedback behavior were obtained in enamel samples for evaluation of topographic changes. In a control experiment, SECM images showed no significant difference in the current monitored during the scan, implying that enamel demineralization did not occur in mineral water medium. Topographic SECM images obtained after contact with citric and hydrochloric acid for different periods of time showed a significant increase in the current relative to a previously protected surface, indicating the structural loss of enamel. The thickness of the enamel layer eroded after contact with hydrochloric acid was significantly higher when compared to the one obtained with citric acid. Hence, our results showed that the enamel acid erosion is a relatively fast process, which is strongly dependent on parameters such as pH, time, acid strength and acid concentration.

In Vitro Analysis of Bacterial Morphology by Atomic Force Microscopy of Low Level Laser Therapy 660, 830 and 904 nm

Objective: The objective of this study was to analyze the bacterial morphology by atomic force microscopy (AFM) after the application of low-level laser therapy (LLLT) in in vitro culture of Staphylococcus aureus ATCC 29213. Background data: Infections caused by S. aureus are among the highest occurring in hospitals and can often colonize pressure ulcers. LLLT is among the methods used to accelerate the healing of ulcers. However, there is no consensus on its effect on bacteria. Materials and methods: After being cultivated and seeded, the cultures were irradiated using wavelengths of 660, 830, and 904 nm at fluences of 0, 1, 2, 3, 4, 5, and 16 J/cm(2). Viable cells of S. aureus strain were counted after 24 h incubation. To analyze the occurrence of morphological changes, the topographical measurement of bacterial cells was analyzed using the AFM. Results: The overall assessment revealed that the laser irradiation reduced the S. aureus growth using 830 and 904 nm wavelengths; the latter with the greatest inhibition of the colony-forming units (CFU/mL) (331.1 +/- 38.19 and 137.38 +/- 21.72). Specifically with 660 nm, the statistical difference occurred only at a fluence of 3 J/cm(2). Topographical analysis showed small changes in morphological conformity of the samples tested. Conclusions: LLLT reduced the growth of S. aureus with 830 and 904 nm wavelengths, particularly with 904 nm at a fluence of 3 J/cm(2), where the greatest topographical changes of the cell structure occurred.

Oocyte meiotic-stage-specific differences in spindle depolymerization in response to temperature changes monitored with polarized field microscopy and immunocytochemistry

Objective: To compare the polymerization status of mouse oocyte spindles exposed to various temperatures at various stages of meiosis. Design: Experimental animal study. Setting: University animal laboratory. Animal(s): CF1 mice. Intervention(s): Immature oocytes matured to metaphase I (MI), telophase I (TI), and metaphase II (MII) were incubated at 37 degrees C (control), room temperature (RT), or 4 degrees C for 0, 10, 30, and 60 minutes. Spindle analysis subsequently was performed using polarized field microscopy and immunocytochemistry. Spindles of TI and MII oocytes that underwent vitrification and warming were analyzed also by immunocytochemistry. Main Outcome Measure(s): Detection of polymerized meiotic spindles. Result(s): At RT, and after 60 minutes at 4 degrees C, a significant time-dependent decrease in the percentage of polymerized meiotic spindles was observed in MI and MII oocytes, but not in TI oocytes. The polymerization of TI spindles at 4 degrees C was similar to that of TI spindles at 4 degrees C that underwent vitrification and warming. Conclusion(s): Significant differences in the microtubule dynamics of MI, TI, and MII oocytes incubated at different temperatures were observed. In particular, meiotic spindles in TI oocytes exhibited less depolymerization than did metaphase spindles. (Fertil Steril (R) 2012; 97: 714-9. (C) 2012 by American Society for Reproductive Medicine.)