Light- and electronmicroscopic study of infiltration of resin into initial caries lesions--a new methodological approach

It was the purpose of this pilot study to investigate resin infiltration into various types of initial subsurface caries lesions using a combined microscopic technique with polarized light microscopy and fluorescence microscopy and subsequent scanning microscopy with EDX-element analysis. Six extracted premolars with initial caries lesions were used. Five were infiltrated with resin after imbibition of the subsurface carious pore volume of enamel with sodium fluorescein solution. After light curing the unbound dye was removed by washing out in water. Serial sections were cut through the lesions and investigated with polarized light microscopy, fluorescence microscopy and simultaneously with both microscopic techniques. The same sections were then studied with scanning electron microscopy and EDX-element analysis to prove the infiltration of the resin into the lesions. The results showed, that the combination technique adds further morphologic information to infiltration behaviour of the resin. The individual volume of early acute lesions versus chronic lesions involving dentin, and the fluorescein bound by resin was well documented in serial sections. The EDX calcium and phosphorus signals correlate negatively with the lesion extension, and the carbon signal correlates positively, thus labelling the resin infiltration. It could be demonstrated that resin infiltration is dependent from the pore volume of the lesion. It can be concluded that the combined polarized light microscopy with fluorescence microscopy is an advantageous tool for studying infiltration of resin into hard tissues.     

Natural enamel caries in polarized light microscopy: differences in histopathological features derived from a qualitative versus a quantitative approach to interpret enamel birefringence

Lesion area measurement of enamel caries using polarized light microscopy (PLM) is currently performed in a large number of studies, but measurements are based mainly on a mislead qualitative interpretation of enamel birefringence in a single immersion medium. Here, five natural enamel caries lesions are analysed by microradiography and in PLM, and the differences in their histopathological features derived from a qualitative versus a quantitative interpretation of enamel birefringence are described. Enamel birefringence in different immersion media (air, water and quinoline) is interpreted by both qualitative and quantitative approaches, the former leading to an underestimation of the depth of enamel caries mainly when the criterion of validating sound enamel as a negatively birefringent area in immersion in water is used (a current common practice in dental research). Procedures to avoid the shortcomings of a qualitative interpretation of enamel birefringence are presented and discussed.     

Microhardness and SEM after CO2 laser irradiation or fluoride treatment in human and bovine enamel

Background: It remains uncertain as to whether or not CO₂ laser is able to hinder demineralization of enamel. The possibility to use bovine instead of human teeth on anticariogenic studies with laser has not yet been determined. Purpose: To compare the ability of CO₂ laser and fluoride to inhibit caries‐like lesions in human enamel and to test whether a similar pattern of response would hold for bovine enamel. Study Design: Ninety‐six enamel slabs (2 × 2 × 4 mm) (48 from bovine and 48 from human teeth) were randomly distributed according to surface treatment (n = 12): CO₂ laser, 5% sodium fluoride varnish (FV), 1.23% acidulated phosphate fluoride (APF) gel, or no treatment (control). Specimens were subjected to a 14‐day in vitro cariogenic challenge. Microhardness (SMH) was measured at 30 μm from the surface. For ultrastructural analysis, additional 20 slabs of each substrate (n = 5) received the same treatment described earlier and were analyzed by SEM. Results: ANOVA and Tukey test ascertained that CO₂ laser promoted the least mineral loss (SMH = 252^a). Treatment with FV resulted in the second highest values (207^b), which was followed by APF (172^c). Untreated specimens performed the worst (154^d). SEM showed no qualitative difference between human and bovine teeth. APF and control groups exhibited surfaces covered by the smear layer. A granulate precipitate were verified on FV group and fusion of enamel crystals were observed on lased‐specimens. Conclusions: CO₂ laser may control caries progression more efficiently than fluoride sources and bovine teeth may be a suitable substitute for human teeth in studies of this nature. Microsc. Res. Tech. 73:1030–1035, 2010. © 2010 Wiley‐Liss, Inc.     

Validation of two dual fluorescence techniques for confocal microscopic visualization of resin penetration into enamel caries lesions

Fluorescence confocal microscopy is a useful tool to analyze the infiltration of enamel caries lesions with low-viscosity resins (infiltrants) in vitro. The conventionally used staining technique, which comprises dye labeling of the resin, has been shown to be limited by chromatographic separation of the resin-dye-mixture during penetration. The aim of this study was to develop an improved dual staining technique and to compare validity and reproducibility of both methods. Human molars with proximal white spots were cut across the demineralizations. After varnishing the cut surfaces, paired lesion halves were infiltrated with an infiltrant using either one of two different staining techniques. For the conventional direct technique (A) the infiltrant was labeled with rhodamine isothiocyanate (RITC) prior to application. Using the new indirect technique (B) lesions were stained with RITC solution and subsequently infiltrated with pure infiltrant. After light curing, unbound dye was bleached by immersion in hydrogen peroxide. Remaining lesion pores were stained with sodium fluorescein solution. Penetration depths (PD) and lesion depths (LD) were evaluated by five examiners using confocal microscopy and compared with the results of scanning electron microscopic (SEM; PD) and microradiographic (TMR; LD) analysis. The indirect technique showed better correlation (intraclass coefficients) with SEM (0.990) and TMR (0.982) compared with the direct technique (SEM: 0.513; TMR: 0.702). Inter- and intrarater reliability was higher for technique B compared with technique A. The new indirect technique yields to more valid and reliable results to visualize infiltrant penetration into natural enamel caries lesions compared with the conventional method. Microsc. Res. Tech. 2009. © 2009 Wiley-Liss, Inc.     

WebSEM: an assessment of K-12 remote microscopy efforts

Within the past 10 years a number of institutions have developed and instituted systems and programs that enable remote control of a scanning electron microscope (SEM). Geared toward use by teachers and educators in K-12 classrooms, these systems have offered access to advanced instrumentation to thousands of students over the past decade. However, despite the enormous potential and promise associated with remote microscopy, the reality has been that most such systems are not utilized to their fullest extent. This is partly due to time constraints on the instrument; many such systems are an integral part of the research and/or teaching focus of the institution that offers the service, and as such, K-12 educators are forced to compete with institutional demands. Often this restricts the amount of lessons that can be conducted to a relatively small number, in rather narrowly defined windows of opportunity. However, even when such constraints do not exist, the number of lessons typically requested remains disappointingly low, and the lessons that are conducted are usually simple examinations lacking in depth. In an effort to determine why the promise of K-12 remote microscopy has not been fully realized, a number of assessments have been carried out at Iowa State University in relation to operation and use of the WebSEM, the Web-controllable SEM operated by the Materials Science and Engineering Department of Iowa State University as a part of Project ExCEL, the Extended Classroom for Enhanced Learning. These assessments indicate that the key to successful use of advanced equipment in K-12 classrooms depends less upon hardware than it does upon local instructional situations. Establishing a personal relationship between the SEM operator and the teacher in the classroom appears to be the best way to increase current use of remote microscopy.     

Comparative authentication of three “snow lotus” herbs by macroscopic and microscopic features

“Snow lotus” is a famous Chinese Materia Medica derived from species of the genus Saussurea (Compositae). To differentiate three representative easily‐confused snow lotus herbs, namely, Saussurea involucrata (Kar. et Kir.) Sch.‐Bip, Saussurea laniceps Hand.‐Mazz., and Saussurea medusa Maxim., macroscopic features of the three herbs were systemically observed, and microscopic features were compared by using ordinary light microscopy, polarized light microscopy and scanning electron microscopy (SEM). The results indicate that, as for macroscopic identification, capitula situation and arrangement, and as for microscopic identification, pollen grains, nonglandular hairs, glandular hairs, and cells of inner surface of the microdiodange can be used to authenticate the three snow lotus herbs. Comprehensive table comparing the characteristics were presented in this study. SEM has been found to provide a number of unique characteristics of pollen grains. Based on the observation of pollen grains, evolution sequence of the three species was speculated. The present method was proven to be efficient, convenient, simple, and reliable, which was successfully applied to the authentication of three snow lotus herbs. Microsc. Res. Tech.1 77:631–641, 2014. © 2014 Wiley Periodicals, Inc.     

In Situ analysis of CO2 laser irradiation on controlling progression of erosive lesions on dental enamel

The present study aimed to evaluate in situ the effect of CO₂ laser irradiation to control the progression of enamel erosive lesions. Fifty‐six slabs of bovine incisors enamel (5 × 3 × 2.5 mm³) were divided in four distinct areas: (1) sound (reference area), (2) initial erosion, (3) treatment (irradiated or nonirradiated with CO₂ laser), (4) final erosion (after in situ phase). The initial erosive challenge was performed with 1% citric acid (pH = 2.3), for 5 min, 2×/day, for 2 days. The slabs were divided in two groups according to surface treatment: irradiated with CO₂ laser (λ = 10.6 µm; 0.5 W) and nonirradiate. After a 2‐day lead‐in period, 14 volunteers wore an intraoral palatal appliance containing two slabs (irradiated and nonirradiated), in two intraoral phases of 5 days each. Following a cross‐over design during the first intraoral phase, half of the volunteers immersed the appliance in 100 mL of citric acid for 5 min, 3×/day, while other half of the volunteers used deionized water (control). The volunteers were crossed over in the second phase. Enamel wear was determined by an optical 3D profilometer. Three‐way ANOVA for repeated measures revealed that there was no significant interaction between erosive challenge and CO₂ laser irradiation (P = 0.419). Erosive challenge significantly increased enamel wear (P = 0.001), regardless whether or not CO₂ laser irradiation was performed. There was no difference in enamel wear between specimens CO₂‐laser irradiated and non‐irradiated (P = 0.513). Under intraoral conditions, CO₂ laser irradiation did not control the progression of erosive lesions in enamel caused by citric acid. Microsc. Res. Tech. 77:586–593, 2014. © 2014 Wiley Periodicals, Inc.     

Morphological and porosity changes in primary enamel surface after an in vitro demineralization model

In vitro models are very useful in dentistry, especially to evaluate preventive methods against dental caries. Although they have been used for more than 30 years, specific demineralization models have not been established for primary enamel, which is more prone to demineralization than permanent enamel. This study evaluates porosity changes in primary enamel surface after a demineralization model through a scientifically validated analytical tool. Nine healthy human anterior primary teeth extracted for therapeutic reasons were included in this study, previous informed consent. The samples were randomly assigned to three groups n = 3: G1_2D, G2_4D, and G3_7D. Scanning electron microscopy (SEM) images at ×200 and ×1000 were taken during two stages: before demineralization (BD) and after demineralization (AD). Morphological characterization was observed at ×1000, while porosity (pore count and perimeter) was analyzed by the ImageJ program, using ×200 SEM images previously converted. Several statistical analyses were used to determine differences (p ≤ .05). Morphological characterization AD revealed new pits and cracks on the enamel surface in G1_2D and G2_4D groups. Localized eroded enamel areas were observed in G3_7D. Pore count of enamel surface BD ranged from 64.26 ± 37.62 to 97.93 ± 34.25 and AD ranged from 150.06 ± 64.86 to 256 ± 58.14. AD, G_4D exhibited a decrease in pore perimeter contrary to G_2D and G_7D. Significant differences were observed. Finally, morphological changes were more evident as days of demineralization increased; 7 days of immersion could be employed as an enamel erosive model. The pore count increased after the demineralization model, BD pores perimeter was heterogeneous, and AD varied according to the immersion period.

• Morphological changes were more evident as days of demineralization increased.
• Seven days of immersion could be employed as an enamel erosive model.
• The initial porosity seems to be a determining factor for the final porousness.
• The pore perimeter of the primary enamel varied according to the immersion period on the demineralization model.

     

Capturing and displaying microscopic images used in medical diagnostics and forensic science using 4K video resolution – an application in higher education

To analyze, interpret and evaluate microscopic images, used in medical diagnostics and forensic science, video images for educational purposes were made with a very high resolution of 4096 × 2160 pixels (4K), which is four times as many pixels as High‐Definition Video (1920 × 1080 pixels). The unprecedented high resolution makes it possible to see details that remain invisible to any other video format. The images of the specimens (blood cells, tissue sections, hair, fibre, etc.) are recorded using a 4K video camera which is attached to a light microscope. After processing, this resulted in very sharp and highly detailed images. This material was then used in education for classroom discussion. Spoken explanation by experts in the field of medical diagnostics and forensic science was also added to the high‐resolution video images to make it suitable for self‐study.     

Ultrastructural changes in the cemento‐enamel junction caused by acidic beverages: An in vitro study

The present in vitro study was aimed at evaluating the morphological changes in the cemento‐enamel junction (CEJ) after exposure to acidic beverages using the scanning electron microscopy (SEM). The initial pH and titratable acidity (TA) was analyzed from follow groups: (I) Coca cola, (II) orange juice, (III) Cedevita, (IV) Red Bull, (V) Somersby cider, and (VI) white wine. The CEJ samples (n = 64), obtained from unerupted third molars, were allocated to one control (artificial saliva, n = 16) and six experimental groups (n = 8). The experimental samples were immersed in beverages (50 ml) for 15 min, three times daily, 10 days, and in artificial saliva between immersions. SEM analysis was performed in a blind manner, according to scoring scale. One‐way ANOVA and Tukey's post hoc tests, as well as Kruskal–Wallis and Mann–Whitney U test used for statistical analysis. The pH values of the acidic beverages ranged from 2.65 (Coca cola) to 3.73 (orange juice), and TA ranged from 1.90 ml (Coca cola) to 5.70 ml (orange juice) of NaOH to reach pH 7.0. The SEM analysis indicated statistically significant differences between the control samples and those immersed in acidic beverages. The Groups IV, I, and II, showed the highest CEJ damage grade while those of the Group VI were the lowest. All the tested acidic beverages caused morphological changes in the CEJ with a smaller or larger exposure of dentine surface, and were not always related to the pH or TA of acidic beverages.     

In vitro evaluation of the microbial contamination on new toothbrushes: a preliminary study

Objective: The presence and survival of microorganisms on toothbrush bristles might play a role on the etiology of oral infections. The aim of this in vitro study was to evaluate the presence of bacterial contamination on new toothbrushes before oral contact. Materials and methods: Forty toothbrushes from five different manufacturers were used in this experimental study. Each manufacturer was divided according to conventional local of obtaining: industry, drugstore, market, and perfumery. The toothbrush heads were completely immersed into tubes containing 5.0 mL of sterile peptonated water (dilution 1:10). A group of eight tubes containing the sterile solution was used as control. After 21 days of anaerobic incubation, occurrence of contamination was visually evaluated and confirmed by light microscopy. Results: Bacterial growth in the medium, indicative of bristles contamination, was found in a total of 19 out of 40 samples (47.5%) evaluated: 6 out of 14 samples (42.85%) from industry group, 4 out of 8 samples (50.0%) from drugstore, 5 out of 10 samples (50.0%) from market, and 4 out of 8 samples (50.0%) from perfumery. Only the toothbrushes with bristles coated with chlorhexidine did not show contamination. The Gram‐negative sporulating bacilli were the most prevalent form recovered. Conclusions: Except for chlorhexidine group, bacterial growth was observed in all groups evaluated irrespective local of obtaining. Microsc. Res. Tech., 2012. © 2011 Wiley Periodicals, Inc.     

High angle [001] twist boundaries in platelet colonies of BiSrCaCuO (2223) superconductor

The microstructure of BiPbSrCaCuO (2223) ceramic was studied by polarised light microscopy and the etch-pits technique. The high-angle twist grain boundaries in the platelet colonies were confirmed to be [001] coincidence type twisted boundaries or rotation twin boundaries with the (001) twinning plane.     

The use of Er:YAG laser for cavity preparation: an SEM evaluation

OBJECTIVE: The purpose of this study was to evaluate morphological changes in cavities prepared by the Er:YAG laser (2.94 mum) at different parameters of irradiation and by a diamond bur. EXPERIMENTAL DESIGN: Cavities were prepared on 27 human molars (n = 3): G1, 15 Hz/160 mJ enamel/6 Hz/200 mJ dentin; G2, 15 Hz/180 mJ enamel/6 Hz/200 mJ dentin; G3, 15 Hz/160 mJ enamel/6 Hz/250 mJ dentin; G4, 15 Hz/180 mJ enamel/6 Hz/250 mJ dentin; G5, 15 Hz/180 mJ enamel/10 Hz/180 mJ dentin; G6, 15 Hz/160 mJ enamel/10 Hz/180 mJ dentin; G7, 15 Hz/160 mJ enamel/10 Hz/160 mJ dentin; G8, 15 Hz/180 mJ enamel/10 Hz/160 mJ dentin; G9, diamond bur. For SEM analysis, samples were fixed (2.5% glutaraldheyde, 12 h, 4 degrees C), dehydrated (25-100% ethanol), dried, and sputter-coated with gold. RESULTS: Despite the changes on energy and repetition-rate settings, all laser-treated samples showed no evidence of thermal damage or signs of burning and melting. Er:YAG laser ablated dental hard tissues showed exposed enamel prisms, dentin surface without smear layer, and opened dentinal tubules. CONCLUSION: Different Er:YAG laser parameters were effective for ablation of hard tissues, creating an irregular and microretentive morphological pattern without hard tissue damage.     

Comparison of photon transport efficiency in simple scintillation electron detector configurations for scanning electron microscope

The purpose of this paper is to find some general rules for the design of robust scintillation electron detectors for a scanning electron microscope (SEM) that possesses an efficient light-guiding (LG) system. The paper offers some general instructions on how to avoid the improper design of highly inefficient LG configurations of the detectors. Attention was paid to the relevant optical properties of the scintillator, light guide, and other components used in the LG part of the scintillation detector. Utilizing the optical properties of the detector components, 3D Monte Carlo (MC) simulations of photon transport efficiency in the simple scintillation detector configurations were performed using the computer application called SCIUNI to assess shapes and dimensions of the LG part of the detector. The results of the simulation of both base-guided signal (BGS) configurations for SE detection and edge-guided signal (EGS) configurations for BSE detection are presented. It is demonstrated that the BGS configuration with a matted disc scintillator exit side connected to the cylindrical light guide without optical cement is almost always a sufficiently efficient system with a mean LG efficiency of about 20%. It is simulated that poorly designed EGS strip configurations have an extremely low mean LG efficiency of only 0.01%, which can significantly reduce detector performance. On the other hand, no simple nonoptimized EGS configuration with a light guide widening to a circular or square profile, with a polished cemented scintillator and with an indispensable hole in it has a mean LG efficiency lower than 6.5%.     

数字显示精测双折射仪的研制与性能分析

介绍了采用光电瞄准的双折射仪的工作原理。从理论上进行了误差分析,并从测试中得到了证实。经使用,该仪器重复性好,可靠性高,测试精度高。     

Different bacterial models for in vitro induction of non‐cavitated enamel caries‐like lesions: Microhardness and polarized light miscroscopy analyses

The aim of this study was to compare different bacterial models for in vitro induction of non‐cavitated enamel caries‐like lesions by microhardness and polarized light microscopy analyses. One hundred blocks of bovine enamel were randomly divided into four groups (n = 25) according to the bacterial model for caries induction: (A) Streptococcus mutans, (B) S. mutans and Lactobacillus acidophilus, (C) S. mutans and L. casei, and (D) S. mutans, L. acidophilus, and L. casei. Within each group, the blocks were randomly divided into five subgroups according to the duration of the period of caries induction (4–20 days). The enamel blocks were immersed in cariogenic solution containing the microorganisms, which was changed every 48 h. Groups C and D presented lower surface hardness values (SMH) and higher area of hardness loss (ΔS) after the cariogenic challenge than groups A and B (P < 0.05). As regards lesion depth, under polarized light microscopy, group A presented significantly lower values, and groups C and D the highest values. Group B showed a higher value than group A (P < 0.05). Groups A and B exhibited subsurface caries lesions after all treatment durations, while groups C and D presented erosion‐type lesions with surface softening. The model using S. mutans, whether or not it was associated with L. acidophilus, was less aggressive and may be used for the induction of non‐cavitated enamel caries‐like lesions. The optimal period for inducing caries‐like lesions was 8 days. Microsc. Res. Tech. 78:444–451, 2015. © 2015 Wiley Periodicals, Inc.     

Correlative light and backscattered electron microscopy of bone--part II: automated image analysis

Detailed studies of biological phenomena often involve multiple microscopy and imaging modes and media. For bone biology, various forms of light and electron microscopy are used to study the microscopic structure of bone. Integrating information from the different sources is necessary to understand how different aspects of the bone structure interact. To accomplish this, methods were developed to prepare and image thin sections for correlative light microscopy (LM) and backscattered electron imaging in the scanning electron microscope (BSE-SEM). Images of the same fields of view may then be analyzed for degrees of relationships between specimen features not observed by LM or SEM alone. These methods are applied here to study possible associations between the degree of bone mineralization and pattern of collagen fiber orientation in the mid-shaft of the human femur. The "relational images" obtained allow us to examine the relationship between these two variables, both objectively and quantitatively.