Porosity variation in hydroxyapatite and osteoblast morphology: a scanning electron microscopy study

The biocompatibility of hydroxyapatite has been demonstrated by previous studies, with enhancement of osteointegration through the use of porous hydroxyapatite (pHA). Emphasis has been focused on the use of coralline hydroxyapatite or the introduction of macroporosity into synthetic hydroxyapatite. The current study investigates the role of macro‐ and microporosities in synthetic phase‐pure porous hydroxyapatite on the morphological aspects of human osteoblast‐like cells using scanning electron microscopy. Cells were seeded on four different types of porous hydroxyapatite (HA1, HA2, HA3 and HA4) and examined following 1, 2, 14 and 30 days of incubation in vitro. The results indicated that the cells had an affinity to micropores through filopodia extensions, at initial stage of attachment. Cellular proliferation and colonization was evident on all materials with cells forming cellular bridges across the macropores at day 14 with cellular canopy formation covering entire macropores observed by day 30. This study demonstrates that while the introduction of microporosity has no evident effect on cellular morphology at later time points, it seems to play a role in initial cellular anchorage and attachment.     

Three‐dimensional morphology of cerebellar climbing fibers. A study by means of confocal laser scanning microscopy and scanning electron microscopy

The intracortical pathway of cerebellar climbing fibers have been traced by means of scanning electron microscpy (SEM) and confocal laser scanning microscopy (CLSM) to study the degree of lateral collateralization of these fibers in the granular Purkinje cell and molecular layers. Samples of teleost fish were processed for conventional and freeze‐fracture SEM. Samples of hamster cerebellum were examined by means of CLSM using FM4–64 as an intracellular stain. High resolution in lens SEM of primate cerebellar cortex was carried out using chromium coating. At scanning electron and confocal laser microscopy levels, the climbing fibers appeared at the white matter and granular layer as fine fibers with a typical arborescence or crossing‐over branching pattern, whereas the mossy fibers exhibited a characteristic dichotomous bifurcation. At the granular layer, the parent climbing fibers and their tendrils collaterals appeared to be surrounding granule and Golgi cells. At the interface between granule and Purkinje cell layers, the climbing fibers were observed giving off three types of collateral processes: those remaining in the granular layer, others approaching the Purkinje cell bodies, and a third type ascending directly to the molecular layer. At this layer, retrograde collaterals were seen descending to the granular layer. By field emission high‐resolution SEM of primate cerebellar cortex, the climbing fiber terminal collaterals were appreciated ending by means of round synaptic knobs upon the spines of secondary and tertiary Purkinje cell dendrites.     

A vision-based, 3D reconstruction technique for scanning electron microscopy: direct comparison with atomic force microscopy

High-resolution, detailed 3D reconstructions of biological specimens obtained from scanning electron microscopy stereo-micrographs and proprietary software were compared with Tapping-Mode AFM datasets of the same fields. The reconstruction software implements several original solutions including a neural adaptive point-matching technique, the ability to build an irregular triangulated mesh rather than a regular orthogonal grid, and the ability to re-map one of the original images exactly onto the reconstructed surface. The technique was applied to human nerve tissue to obtain 1,424 x 968-pixel, texture-mapped datasets, which were subsequently compared against 512 x 512-pixel AFM datasets from the same viewfields. Accounting for the inherent differences of the two techniques, direct comparison revealed an excellent visual match. The correspondence was also quantified by calculating the cross-correlation coefficient between corresponding altimetric profiles in SEM and AFM data, which consistently exceeded a figure of 0.9, with a rate of point mismatch in the order of 0.01%. Research is still underway to improve the robustness of the technique when applied to arbitrary images     

Sample holder for axial rotation of specimens in 3D microscopy

In common light microscopy, observation of samples is only possible from one perspective. However, especially for larger three‐dimensional specimens observation from different views is desirable. Therefore, we are presenting a sample holder permitting rotation of the specimen around an axis perpendicular to the light path of the microscope. Thus, images can be put into a defined multidimensional context, enabling reliable three‐dimensional reconstructions. The device can be easily adapted to a great variety of common light microscopes and is suitable for various applications in science, education and industry, where the observation of three‐dimensional specimens is essential. Fluorescence z‐projection images of copepods and ixodidae ticks at different rotation angles obtained by confocal laser scanning microscopy and light sheet fluorescence microscopy are reported as representative results.     

Adverse effects of respiratory disease medicaments and tooth brushing on teeth: A scanning electron microscopy,X‐ray fluorescence and infrared spectroscopy study

The present study aims to evaluate the effect of brushing with fluoride dentifrice on teeth severely affected by erosion due to respiratory medicaments. Enamel (n = 50) and dentin (n = 50) bovine specimens were prepared and treated with artificial saliva (S‐control), acebrofilin hydrochloride (AC), ambroxol hydrochloride (AM), bromhexine hydrochloride (BR), and salbutamol sulfate (SS) and subjected to cycles of demineralization (immersing in 3 mL, 1 min, three times a day at intervals of 1 hr, for 5 days) followed by remineralization (saliva, 37°C, 1 hr). Simulated brushing with fluoridated toothpaste was performed using 810 strokes in a reciprocal‐action brushing simulator. Scanning electron microscopy, micro energy dispersive X‐ray fluorescence (μ‐EDXRF) spectroscopy and attenuated total reflection Fourier transform infrared (ATR FTIR) spectroscopy were then performed. μ‐EDXRF images showed extensive erosion after treatment with all medicaments. SEM images showed enamel erosion in order SS > BR > AC = AM > S after brushing and fluoridation. FTIR results were in agreement. In case of dentin, μ‐EDXRF measurements showed significant difference in mineral content (percent weight of calcium and phosphate) in SS + brushing + fluoridation treated enamel compared to control, while μ‐EDXRF images showed erosive effects in the order SS > AM>BR > AC = S post brushing + fluoridation. SEM images showed erosion in the order SS > AM = BR > AC > S post brushing + fluoridation. Again, FTIR multivariate results were in agreement. Overall, our study shows that proper oral care is critical when taking certain medication. The study also demonstrates the possible use of FTIR for rapid clinical monitoring of tooth erosion in clinics.     

Algorithms for accurate 3D registration of neuronal images acquired by confocal scanning laser microscopy

This paper presents automated and accurate algorithms based on high‐order transformation models for registering three‐dimensional (3D) confocal images of dye‐injected neurons. The algorithms improve upon prior methods in several ways, and meet the more stringent image registration needs of applications such as two‐view attenuation correction recently developed by us. First, they achieve high accuracy (≈ 1.2 voxels, equivalent to 0.4 µm) by using landmarks, rather than intensity correlations, and by using a high‐dimensional affine and quadratic transformation model that accounts for 3D translation, rotation, non‐isotropic scaling, modest curvature of field, distortions and mechanical inconsistencies introduced by the imaging system. Second, they use a hierarchy of models and iterative algorithms to eliminate potential instabilities. Third, they incorporate robust statistical methods to achieve accurate registration in the face of inaccurate and missing landmarks. Fourth, they are fully automated, even estimating the initial registration from the extracted landmarks. Finally, they are computationally efficient, taking less than a minute on a 900‐MHz Pentium III computer for registering two images roughly 70 MB in size. The registration errors represent a combination of modelling, estimation, discretization and neuron tracing errors. Accurate 3D montaging is described; the algorithms have broader applicability to images of vasculature, and other structures with distinctive point, line and surface landmarks.     

Polymer-based materials to be used as the active element in microsensors: a scanning force microscopy study

Polymer-based materials can be incorporated as the active sensing elements in chemiresistor devices. Most of these devices take advantage of the fact that certain polymers will swell when exposed to gaseous analytes. To measure this response, a conducting material such as carbon black is incorporated within the nonconducting polymer matrix. In response to analytes, polymer swelling results in a measurable change in the conductivity of the polymer/carbon composite material. Arrays of these sensors may be used in conjunction with pattern recognition techniques for purposes of analyte recognition and quantification. We have used the technique of scanning force microscopy (SFM) to investigate microstructural changes in carbon-polymer composites formed from the polymers poly (isobutylene) (PIB), poly (vinyl alcohol) (PVA), and poly (ethylene-vinyl acetate) (PEVA) when exposed to the analytes hexane, toluene, water, ethanol, and acetone. Using phase-contrast imaging (PI), changes in the carbon nanoparticle distribution on the surface of the polymer matrix are measured as the polymers are exposed to the analytes in vapor phase. In some but not all cases, the changes were reversible (at the scale of the SFM measurements) upon removal of the analyte vapor. In this paper, we also describe a new type of microsensor based on piezoresistive microcantilever technology. With these new devices, polymeric volume changes accompanying exposure to analyte vapor are measured directly by a piezoresistive microcantilever in direct contact with the polymer. These devices may offer a number of advantages over standard chemiresistor-based sensors.     

Penetration of resin‐based materials into initial erosion lesion: A confocal microscopic study

The application of resin‐based materials is an alternative of treatment for eroded lesions. Nevertheless, there are no studies about the penetration of these materials into eroded lesion, which might affect its adhesion. Therefore, this study evaluated the penetration of four resin‐based materials, with and without enamel etching. By using an in vitro protocol, types of treatment were studied at five levels (AdheSE^®, Tetric N‐Bond^®, Single Bond 2^®, Helioseal Clear^®, Icon^®) and types of enamel etching in two levels (with and without). Materials were stained with 0.02 mg/mL ethanolic solution of tetramethylrhodamine isothiocyanate. Bovine enamel samples (4 × 4 mm) were immersed in 0.01 M HCl, pH 2.3, for 30 seconds to produce initial eroded lesions. Afterward, the materials were applied on half of sample enamel surface following the manufacturer's instructions. On the other half of sample, the materials were applied without etching the enamel. Materials penetration into the enamel was assessed by Confocal Laser Scanning Microscopy on reflection and fluorescence modes. The penetration depth (PD) was measured using ImageJ software. Data were analyzed by two‐way ANOVA and Tukey test (P < 0.05). Regardless of the material, etched enamel resulted in higher PD than non‐etched (P < 0.05). Icon^® showed the highest PD in enamel followed by Helioseal Clear^® (P < 0.05), with significant difference between them (P < 0.05) and no difference was found among AdheSE^®, Tetric N‐Bond^®, and Single Bond 2^® (P > 0.05). It can be concluded that prior enamel etching increased the materials penetration into eroded enamel and the Icon^®—infiltrant presented highest penetration. Microsc. Res. Tech. 79:72–80, 2016. © 2015 Wiley Periodicals, Inc.     

Confocal laser scanning microscopy to study formation and properties of polyelectrolyte nanocapsules derived from CdCO3 templates

Three-dimensional confocal laser scanning microscopy (CLSM) was used as an essential investigation method to obtain information about the formation and morphological characteristics of nanocapsules. Nanocapsules are built by layer-by-layer deposition of alternatively charged polyelectrolytes on templates forming nanostructured hollow shells. CLSM is unique in allowing for monitoring of the core dissolution process in real time and for studying nanocapsule functioning in hydrated conditions within a three-dimensional and temporal framework. Since we are also interested in the identification of other possible templates, we briefly report on the use of yeast cells as biocolloidal cores monitored by means of two-photon microscopy. Here we focus our attention on the use of CdCO(3) crystals as template candidates for the preparation of stable capsules. Both cubic and spherical CdCO(3) cores have been produced. Cubic cores exhibit higher monodispersity and smaller size compared to spherical ones. Capsules templated on these cores have a higher surface-to-volume ratio that is valuable for applications related to drug delivery, functional properties of the shells and adsorption of proteins, and other biologically relevant molecules. Microsc. Res. Tech. 59:536-541, 2002.     

Measurements of fillet weld by 3D laser scanning system

The external geometry of a fillet weld affects not only the quality but also the safety of a structure. Two parameters that influence the geometric quality of a fillet weld are the weld size and the weld surface profile. A 3D laser scanning measurement system integrating the techniques of reverse engineering is adopted in this study to provide a more accurate measurement of the weld profile. The section profiles of the external weld geometry along the longitudinal axis of the weld are generated by the measured data set through CAD software. A computer programme based on the AWS Structural Welding Code is used to judge the acceptability of a fillet weld based on the CAD section profile of the weld. The proposed 3D laser scanning system with integrated CAD software can provide a more accurate and efficient method to estimate the geometric quality of a fillet weld .     

Tubular dentin sealer penetration after different final irrigation protocols: A confocal laser scanning microscopy study

The aim of this study was to evaluate tubular dentin sealer penetration, comparing different final irrigation protocols using a conventional needle (CONV), EndoActivator system (EAS), EndoVac system (EVS), and ultrasound (PUI). Initially, fifty‐two first maxillary molars with a single canal in the palatal root, without abrupt curvatures, resorptive processes, or previous endodontic treatment were selected for this study. Then, the crowns were sectioned to obtain palatal roots 15 mm in length. The root canals were prepared with the ProTaper Universal System and irrigated with 5% NaOCl. Afterwards, the specimens were divided into four groups (n. 13), according to the final irrigation protocol: CONV, EAS, EVS, and PUI. After filling, slices at 3 mm and 5 mm from the apex were obtained for analysis by confocal laser scanning microscopy. Two‐way comparisons between the groups and the levels were performed with Games Howell's test (p < .05). Tubular dentin sealer penetration was higher at 5 mm compared with 3 mm from the apex (p < .05). The EAS group showed a higher percentage of tubular dentin sealer penetration, compared with the CONV group, at both levels. At 3 mm, there was no statistically significant difference among EAS, EVS, and PUI; however, these groups showed better performance, compared with the CONV group. At 5 mm, there was no statistically significant difference between the EAS and EVS groups, but both showed higher sealer penetration than the PUI group (p < .05). The EAS and EVS groups achieved better degrees of tubular dentin sealer penetration, compared with the other groups.     

Quantitative tomography of early mouse embryos: laser scanning microscopy and 3D reconstruction

The cell volume alteration participates in a wide variety of cellular functions that may interfere with intra‐cellular homeostasis. The most adequate approach of estimation of the volume changes induced by osmotic misbalance, alteration in shape and size due to the action shape forming substances, etc., is the direct measurement of volumetric parameters of embryos. In the given research, the volume magnitude and kinetics of changes in volume and surface area of blastomere and polar bodies of early mouse embryos were determined using three‐dimensional reconstruction of the optical section stack obtained with laser scanning microscope (LSM). The size and surface area were determined for isotonic and anisosmotic conditions. The physiological significance of the findings is discussed.     

New data‐driven method from 3D confocal microscopy for calculating phytoplankton cell biovolume

Confocal laser scanner microscopy coupled with an image analysis system was used to directly determine the shape and calculate the biovolume of phytoplankton organisms by constructing 3D models of cells. The study was performed on Biceratium furca (Ehrenberg) Vanhoeffen, which is one of the most complex‐shaped phytoplankton. Traditionally, biovolume is obtained from a standardized set of geometric models based on linear dimensions measured by light microscopy. However, especially in the case of complex‐shaped cells, biovolume is affected by very large errors associated with the numerous manual measurements that this entails. We evaluate the accuracy of these traditional methods by comparing the results obtained using geometric models with direct biovolume measurement by image analysis. Our results show cell biovolume measurement based on decomposition into simple geometrical shapes can be highly inaccurate. Although we assume that the most accurate cell shape is obtained by 3D direct biovolume measurement, which is based on voxel counting, the intrinsic uncertainty of this method is explored and assessed. Finally, we implement a data‐driven formula‐based approach to the calculation of biovolume of this complex‐shaped organism. On one hand, the model is obtained from 3D direct calculation. On the other hand, it is based on just two linear dimensions which can easily be measured by hand. This approach has already been used for investigating the complexities of morphology and for determining the 3D structure of cells. It could also represent a novel way to generalize scaling laws for biovolume calculation.     

A method of direct particle deposition on a carbon planchet to study mineral dust in human lung by scanning electron microscopy

Following Na-hypochlorite digestion of lung tissue, mineral particles extracted in the chloroform layer were deposited directly on a pre-smoothed carbon planchet for combined scanning electron microscopy and X-ray energy dispersive spectrometry (SEM and XEDS). Total mineral particle counts were obtained, and detailed physical characteristics of the fibrous particles were documented at 600, 1,500, 4,500 and 9,000 x in three lungs without, and one lung with, histories of occupational exposure. This preparation method was simple, collected more than 99% of identifiable mineral particles in the chloroform layer, gave excellent object to background contrast without heavy metal coatings, and was suitable for XEDS. Comparable fibrous particles from the chloroform layer could also be studied by selected-area electron diffraction to complement the results of XEDS. By this method, we found particles or fibers larger than 0.1 μm were readily counted and measured at 4,500 x. At 600 x, ferruginous bodies were found to be more than twice in number than when sought for by light microscopy. It was determined that 4,500 x is the most efficient magnification to examine and diagnose this type of specimen. The present study illustrates the importance of determining the most efficient magnification to be utilized in particle counts.     

3‐D PSF fitting for fluorescence microscopy: implementation and localization application

Localization microscopy relies on computationally efficient Gaussian approximations of the point spread function for the calculation of fluorophore positions. Theoretical predictions show that under specific experimental conditions, localization accuracy is significantly improved when the localization is performed using a more realistic model. Here, we show how this can be achieved by considering three‐dimensional (3‐D) point spread function models for the wide field microscope. We introduce a least‐squares point spread function fitting framework that utilizes the Gibson and Lanni model and propose a computationally efficient way for evaluating its derivative functions. We demonstrate the usefulness of the proposed approach with algorithms for particle localization and defocus estimation, both implemented as plugins for ImageJ.