Influence of calcitonin administration on ultimobranchial and parathyroid glands of pigeon,Columba livia

Pigeons were divided into two numerically equal groups (A and B) containing 30 specimens each. Birds from group A were intraperitoneally injected daily with 0.1 mL/100 g body wt of vehicle (0.1 M acetate buffer, pH 4.3 containing 0.1% gelatin). Specimens from group B were intraperitoneally injected daily with 1 μg/100 g body wt of salmon calcitonin. Six birds were sacrificed from each group 2 h after the last injection on 1st, 3rd, 5th, 10th, and 15th day of the experiment. After collection of blood samples, ultimobranchial and parathyroid glands were fixed for histological studies. In calcitonin-treated C. livia, the plasma calcium levels exhibit a progressive decline from day 1 till day 5. On day 15, the levels become more or less similar to the control value. No change has been noticed on day 1 in the plasma phosphate levels of calcitonin-treated C. livia. The levels decrease progressively from day 3 to day 5; thereafter, it exhibits an elevation so that on day 15, normal plasma phosphate levels is achieved. The ultimobranchial gland of C. livia exhibits no change up to day 5 following calcitonin treatment. The nuclear volume of ultimobranchial cells exhibits a decrease on day 10. This response progresses up to day 15. Few degenerating cells are also discerned following 15 days calcitonin treatment. The parathyroid gland of calcitonin-treated C. livia exhibits no histological alteration up to day 3. The nuclear volume of parathyroidal cells exhibits a progressive increase from day 3 till the close of the experiment (day 15). Moreover, the gland exhibits more compactness on day 10 and day 15. Few degenerating cells are encountered after day 15 following calcitonin treatment. Microsc. Res. Tech. 2009. © 2008 Wiley-Liss, Inc.     

Response of serum minerals (calcium,phosphate, and magnesium) and endocrine glands (calcitonin cells and parathyroid gland) of wistar rat after chlorpyrifos administration

Wistar rats (male) were daily administered chlorpyrifos at a dose of 5 mg/kg b wt. and 10 mg/kg b wt. and sacrificed on 1st, 2nd, 4th, 6th, and 8th week. In chlorpyrifos exposed rats hypocalcemia, hypophosphatemia and hypomagnesemia were recorded. At later intervals an increased levels of serum calcium and phosphate were observed. The parathyroid glands and calcitonin cells exhibited increased activity which is evident by increased nuclear volume of these cells. Microsc. Res. Tech. 76:673–678, 2013. © 2013 Wiley Periodicals, Inc.     

Topology of the mammalian cell via cryo-FIB etching

Mapping of a mammalian cell down to a feature size of 20–30 nm in 3D is a goal that will answer many questions concerning the connectivity (topology) of a Eukaryotic cell's traffic routes. These routes are defined and separated from one another by the protein-impregnated lipid membrane barrier of the endoplasmic reticulum (ER). We trace the routes from outside a live flash frozen buccal epithelial cell via gold (Au) labelled pores in the plasma membrane to the ER below and then through the cell as isosurfaces in 3D maps. The outer tubular ER with three-way branching changes to a sheet-like ER nearer the nucleus, and the cytoplasmic space between the ER membranes continues as a volume into the nuclear interior via the nuclear pores. We find some evidence that the last layer of the cytoplasmic ER membrane, also termed the outer nuclear membrane, has discrete gaps, so the ER lumen in these areas is continuous with the nuclear luminal domain and further, the inner nuclear membrane has small protrusions into the nucleus. The routes were established in live, unstained, unfixed, cells etched with a pAmp current of a focused ion beam (cryo-FIB) dual beam electron microscope, at −150°C, 1e-4Pa, and confirmed at 37°C in lipid-dye stained cells. The cryo-FIB etch of a cuboid of 2D planes, and its reconstruction into many 3D maps, takes only hours, facilitating the execution of experiments with comparative conditions in a few days.     

Three‐dimensional morphometric comparison of normal and apoptotic endothelial cells based on laser scanning confocal microscopy observation

Three‐dimensional (3D) morphometric analysis of cellular and subcellular structures provides an effective method for spatial cell biology. Here, 3D cellular and nuclear morphologies are reconstructed to quantify and compare morphometric differences between normal and apoptotic endothelial cells. Human umbilical vein endothelial cells (HUVECs) are treated with 60 μM H₂O₂ to get apoptotic cell model and then a series of sectional images are acquired from laser scanning confocal microscopy. The 3D cell model containing plasma membrane and cell nucleus is reconstructed and fused utilizing three sequential softwares or packages (Mimics, Geomagic, and VTK). The results reveal that H₂O₂ can induce apoptosis effectively by regulating the activity of apoptosis‐related biomolecules, including pro‐apoptotic factors p53 and Bax, and anti‐apoptotic factor Bcl‐2. Compared with the normal HUVECs, the apoptotic cells exhibit significant 3D morphometric parameters (height, volume and nucleus‐to‐cytoplasm ratio) variation. The present research provides a new perspective on comparative quantitative analysis associated with cell apoptosis and points to the value of LSCM as an objective tool for 3D cell reconstruction. Microsc. Res. Tech. 76:1154–1162, 2013. © 2013 Wiley Periodicals, Inc.     

Combining quantitative 2D and 3D image analysis in the serial block face SEM: application to secretory organelles of pancreatic islet cells

A combination of two‐dimensional (2D) and three‐dimensional (3D) analyses of tissue volume ultrastructure acquired by serial block face scanning electron microscopy can greatly shorten the time required to obtain quantitative information from big data sets that contain many billions of voxels. Thus, to analyse the number of organelles of a specific type, or the total volume enclosed by a population of organelles within a cell, it is possible to estimate the number density or volume fraction of that organelle using a stereological approach to analyse randomly selected 2D block face views through the cells, and to combine such estimates with precise measurement of 3D cell volumes by delineating the plasma membrane in successive block face images. The validity of such an approach can be easily tested since the entire 3D tissue volume is available in the serial block face scanning electron microscopy data set. We have applied this hybrid 3D/2D technique to determine the number of secretory granules in the endocrine α and β cells of mouse pancreatic islets of Langerhans, and have been able to estimate the total insulin content of a β cell.     

Involvement of the adrenal glands and testis in gap junction formation via testosterone within the male rat anterior pituitary gland

We investigated the influence of testicular and adrenal androgens on the presence of gap junctions between folliculo‐stellate cells in the anterior pituitary glands of 60‐day‐old Wistar‐Imamichi strain male rats. The animals were separated into six groups: Group A served as the controls and had free access to a normal diet and water, Group B was given a normal diet and 0.9% NaCl for their drinking water as the controls of adrenalectomized groups, Group C was castrated, Group D was adrenalectomized, Group E was both castrated and adrenalectomized, and Group F was also both castrated and adrenalectomized. In addition, the animals of Group F were administered a dose of testosterone that is known to produce high physiological levels of the hormones in plasma. Five rats from each group were sacrificed 1, 2, 3, 4, 5, 6, and 7 days after their respective operation, and the anterior pituitary glands were removed and prepared for observation by transmission electron microscopy. We quantified the number of follicles and gap junctions and calculated the rate of occurrence as the ratio of the number of gap junctions existing between folliculo‐stellate cells per intersected follicle profile. Simultaneous removal of adrenal glands with castration resulted in a significantly decrease in the number of gap junctions, whereas the administration of testosterone to these rats compensated for this change. These observations indicate that the preservation of gap junctions between folliculo‐stellate cells is mainly dependent on androgens from both the testes and adrenal glands in adult male rats. Microsc. Res. Tech., 2012. © 2012 Wiley Periodicals, Inc.     

3D map distribution of metallic nanoparticles in whole cells using MeV ion microscopy

In this work, a new tool was developed, the MORIA program that readily translates Rutherford backscattering spectrometry (RBS) output data into visual information, creating a display of the distribution of elements in a true three‐dimensional (3D) environment. The program methodology is illustrated with the analysis of yeast Saccharomyces cerevisiae cells, exposed to copper oxide nanoparticles (CuO‐NP) and HeLa cells in the presence of gold nanoparticles (Au‐NP), using different beam species, energies and nuclear microscopy systems. Results demonstrate that for both cell types, the NP internalization can be clearly perceived. The 3D models of the distribution of CuO‐NP in S. cerevisiae cells indicate the nonuniform distribution of NP in the cellular environment and a relevant confinement of CuO‐NP to the cell wall. This suggests the impenetrability of certain cellular organelles or compartments for NP. By contrast, using a high‐resolution ion beam system, discretized agglomerates of Au‐NP were visualized inside the HeLa cell. This is consistent with the mechanism of entry of these NPs in the cellular space by endocytosis enclosed in endosomal vesicles. This approach shows RBS to be a powerful imaging technique assigning to nuclear microscopy unparalleled potential to assess nanoparticle distribution inside the cellular volume.     

Far-field fluorescence microscopy with three-dimensional resolution in the 100-nm range

We report three-dimensional (3D) microscopy with nearly isotropic resolution in the λ/5 − λ/10 range. Our approach combines 4Pi-confocal two-photon fluorescence microscopy with image restoration. The 3D resolution is demonstrated with densely clustered beads as well as with F-actin fibers in mouse fibroblast cells. A comparison with unrestored two-photon confocal images reveals a total reduction of the uncertainty volume up to a factor of 15.     

Carotid body and glomus cells distributed in the wall of the common carotid artery in the bird

In the bird the carotid body is located between the distal (nodose) ganglion of the vagus nerve and the recurrent laryngeal nerve at the beginning of the common carotid artery, that is, the organ is located at the cervicothoracic border. The chicken carotid body receives numerous branches from the vagus and the recurrent laryngeal nerves. In addition, dense networks of the peptidergic nerve fibers immunoreactive for substance P, calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP), galanin, and neuropeptide Y (NPY) are distributed in and around the carotid body parenchyma. The substance P- and CGRP-immunoreactive fibers are derived from both the superior and inferior ganglia of the vagus nerve. The VIP-, galanin-, and NPY-immunoreactive fibers originate from the 14th cervical ganglion of the sympathetic trunk. The endocrine organs including the thyroid gland, parathyroid glands, carotid body, and ultimobranchial gland are situated as a continuous series along the common carotid artery. The organs are supplied with arteries arising as one trunk from the common carotid artery. Glomus cells are widely distributed not only in the carotid body but also in the wall of the common carotid artery and around the common trunk and its branches. The glomus cells of the chicken carotid body exhibit intense immunoreactivity for serotonin, tyrosine hydroxylase, and chromogranin A. The cells located in the wall of the common carotid artery further express NPY mRNA and peptide. In the chickens exposed to isocapnic hypoxia for 35 days, 3-4-fold increase of the carotid body volume is induced and the carotid body glomus cells show enhanced synthetic and secretory activities. On the other hand, the cells in the wall of the common carotid artery display little changes after the long-term hypoxia, having different functions from the carotid body. The carotid body rudiment is formed in the lateral wall of the third branchial artery. The neural cells immunoreactive for TuJ1, PGP 9.5, and HNK-1, which are continuous with the inferior vagal (nodose) ganglion, first surround and then invade both the carotid body rudiment and the other portions of the third branchial artery, becoming glomus cells.     

Dentin erosion by whitening mouthwash associated to toothbrushing abrasion: A focus variation 3D scanning microscopy study

The aim of this study was to determine the erosive potential of hydrogen peroxide (HP) containing mouthwash on dentin assessed by Focus variation three‐dimensional (3D) microscopy. Twenty dentin slabs were selected and randomly allocated into two groups (n = 10): DW—Distilled water (pH = 7.27) and HP—1.5% (pH = 3.78). Each specimen was cyclically demineralized (4 × 60 s/day, 10 days) with HP or DW and brushed 3×/day (200 g, 150 strokes—toothpaste with 1,450 ppmF as NaF). Between the challenges, the specimens were exposed to artificial saliva. Afterward, dentin loss was analyzed using focus variation 3D microscopy, and the data were submitted to unpaired t‐test (α = 0.05). Statistically significant difference was found between the mean wear rate (μm, ±SD) of HP (1.98 ± 0.51) and DW (1.45 ± 0.39). The results suggest that the use of HP‐containing mouthwash associated to brushing may increase the risk of tissue loss and focus variation 3D microscopy may be used as a technique for quantifying dental wear. Microsc. Res. Tech. 76:904–908, 2013. © 2013 Wiley Periodicals, Inc.     

Residues of calcium hydroxide-based intracanal medication associated with different vehicles: a scanning electron microscopy evaluation

This study evaluated the presence of residues after removal of calcium hydroxide [Ca(OH)(2) ] associated with different vehicles. Thirty single-rooted teeth were instrumented to a master apical file #25 using 2.5% NaOCl as main irrigant and 17% trisodium EDTA (ethylenediaminetetraacetic acid) as final agent irrigant. Then, the root canals were dressed with Ca(OH)(2) associated with silicone oil (Group 1), 2% chlorhexidine gluconate (Group 2), or propylene glycol (Group 3). After coronal sealing, all teeth were kept in a moist environment at room temperature. After 7 days, the teeth were reopened and medicaments were removed using 5 mL of saline solution and instrumentation with master apical file followed by new irrigation with 5 mL of 2.5% NaOCl. Subsequently, teeth were split longitudinally and assessed by scanning electron microscopy. The wall cleanliness of the cervical and apical thirds of the roots were evaluated and scored by three blinded examiners. Statistical analysis was performed using Kruskal-Wallis and Wilcoxon tests at 5% level of significance. All roots had residues of Ca(OH)(2) on the canal walls. All experimental groups had similar results (P > 0.05) regardless of the third evaluated. There was significant difference between the apical and cervical thirds only in Group 3 (P < 0.05). Association of different vehicles to Ca(OH)(2) does not influence the persistence of residues on the root canal walls.     

Spatial control of pa-GFP photoactivation in living cells

Photoactivatable green fluorescent protein (paGFP) exhibits peculiar photo-physical properties making it an invaluable tool for protein/cell tracking in living cells/organisms. paGFP is normally excited in the violet range (405 nm), with an emission peak centred at 520 nm. Absorption cross-section at 488 nm is low in the not-activated form. However, when irradiated with high-energy fluxes at 405 nm, the protein shows a dramatic change in its absorption spectra becoming efficiently excitable at 488 nm. Confocal microscopes allow to control activation in the focal plane. Unfortunately, irradiation extends to the entire illumination volume, making impracticable to limit the process in the 3D (three-dimensional) space. In order to confine the process, we used two advanced intrinsically 3D confined optical methods, namely: total internal reflection fluorescence (TIRF) and two-photon excitation fluorescence (2PE) microscopy. TIRF allows for spatially selected excitation of fluorescent molecules within a thin region at interfaces, i.e. cellular membranes. Optimization of the TIRF optical set-up allowed us to demonstrate photoactivation of paGFP fused to different membrane localizing proteins. Exploitation of the penetration depth showed that activation is efficiently 3D confined even if limited at the interface. 2PE microscopy overcomes both the extended excitation volume of the confocal case and the TIRF constraint of operating at interfaces, providing optical confinement at any focal plane in the specimen within subfemtoliter volumes. The presented results emphasize how photoactivation by non-linear excitation can provide a tool to increase contrast in widefield and confocal cellular imaging.     

不同纳米添加剂下GCr15/45钢自修复性能研究

利用高精度液压式往复试验机研究了纳米羟基磷酸钙、纳米二氧化钛、纳米氮化钛3种纳米添加剂润滑条件下GCr15/45钢对摩时的摩擦磨损性能,通过扫描电子显微镜和EDX能谱对磨斑进行了微观分析。结果表明:纳米润滑添加剂可以降低摩擦副摩擦因数和材料磨损量,表现出优良的抗磨损性能;3种纳米添加剂具有不同的自修复机制,其中纳米羟基磷酸钙和纳米二氧化钛的修复机制主要为铺展成膜自修复,而纳米氮化钛为铺展成膜自修复和原位摩擦化学自修复并存;纳米氮化钛的自修复效果最佳,纳米二氧化钛的自修复性能最差。     

Noninvasive 3D vital imaging and characterization of notochordal cells of the intervertebral disc by femtosecond near-infrared two-photon laser scanning microscopy and spatial-volume rendering

The central region of the intervertebral disc (IVD) in infant humans is made and maintained by notochordal cells (NCs). These cells disappear during maturation to be replaced by mature chondrocyte-like cells. NCs are completely different morphologically from the mature chondrocyte-like IVD cells and have complex and essential functions but little is known about them. Recently, two-photon laser scanning microscopy (TPLSM) using near-infrared (NIR) femtosecond pulsed lasers has emerged as a promising noninvasive optical technique for observing unfixed living 3D biological specimens in situ and in vitro. Several lines of evidence suggest that compared with conventional laser scanning confocal microscopy (LSCM), femtosecond NIR laser-based TPLSM has any number of advantages including 3D resolution without a spatial filter (confocal pinhole), minimal photobleaching, and photodamage above and below the focal plane, and importantly, greater depth penetration. We have thus taken advantage of these unique features of femtosecond laser-based TPLSM for vital 3D imaging in conjunction with advanced spatial-volume rendering modalities to compare morphologies of NCs/clusters from pig caudal discs with chondrocyte-like IVD cells from bovine caudal discs, both in ex vivo tissue and when isolated and grown in vitro within 3D alginate scaffolds. Our results provide evidence that (a) ex vivo notochordal tissue consists of areas with NC clusters, and those dominated by tubular structures of low cell density (b) within 3D in vitro scaffolds the morphology of NC is heterogeneous and the cells contain distinct cytoplasmic vacuole-like structures occasionally including acidic subinclusions (c) a quantitative determination based on 3D spatial and volumetric-rendering reveals an average NC diameter of 22.05 microm (range 11.96-46.63 microm) and NC volume of 9701 microm(3) (2041-36427 microm(3)) whereas chondrocyte-like cells have a mean volume of 3279 microm(3) and diameter of 12.20 microm. Taken together, this study demonstrates that femtosecond TPLSM has unique advantages over other conventional histological and in particular LSCM for high resolution noninvasive vital characterization of notochordal and chondrocyte-like cells of IVD over extended depths beyond 300-500 microm.     

Computer reconstruction of nucleolar architecture by interactive three-dimensional color display

The first complete three dimensional ultrastructural reconstruction of pancreatic cell nucleoli, was done using EM and computer 3D-assisted reconstruction of serial sections with interactive 3D back-to-front and color display methods based on voxel representation. The purpose of the study was to depict the architecture of the nucleolar components. We obtained information about the location of the nucleolus within the nuclear volume and about the shape and polarity of the 3 main nucleolar territories.     

Plasma enclosed in a magnetic field produced by flexible surface magnets

A homogeneous steady state plasma with a usable volume of approximately 200 l and with an electron temperature of 1-2 eV and a plasma density of approximately 10(9)-10(10) cm(-3) is produced in a discharge chamber the outside of whose walls is covered with flexible magnetic strips. This magnet arrangement can be built at a fraction of the cost of a conventional system using rigid surface magnets. The magnetic multipole field leads to an increase of the plasma density by one to two orders of magnitude and it is also found to cause trapping of high energy electrons originating from the discharge region.     

粉末X射线衍射分析法鉴定乙烯球罐外输泵出口过滤网堵塞物样品中晶体物相

采用粉末X射线衍射(PXRD)分析法鉴定了乙烯球罐外输泵出口过滤网堵塞物样品中晶体物相。相鉴定结果为:Fe2O3(赤铁矿PDF#33-0664),Fe+3O(OH)(针铁矿PDF#29-0713),Fe+3O(OH)(纤铁矿PDF#44-1415),CaCO3(方解PDF#05-0586)和SiO2(石英PDF#46-1045)。目视检测发现,堵塞物样品中还含有大量纤维。本试验对乙烯球罐外输泵出口过滤网堵塞故障技术分析与技改具有指导意义。     

High speed laser tomography system

A high speed laser tomography system was developed capable of acquiring three-dimensional (3D) images of optically thin clouds of moving micron-sized particles. It operates by parallel-shifting an illuminating laser sheet with a pair of galvanometer-driven mirrors and synchronously recording two-dimensional (2D) images of thin slices of the imaged volume. The maximum scanning speed achieved was 120,000 slices/s, sequences of 24 volume scans (up to 256 slices each) have been obtained. The 2D slices were stacked to form 3D images of the volume, then the positions of the particles were identified and followed in the consecutive scans. The system was used to image a complex plasma with particles moving at speeds up to cm/s.     

Charge and charging compensation on oxides and hydroxides in oxygen environmental SEM

Oxygen environment was applied to the scanning electron microscopy (SEM) analysis of insulating samples. In the high vacuum SEM, a local oxygen pressure was provided, and in the environmental SEM, oxygen atmosphere was used instead of water, the commercial mode. The charging effects in the SEM observation and component characterization of samples such as Al(2)O(3), Al(OH)(3), Mg(OH)(2) and others can be eliminated or significantly reduced. The oxygen environment does not only provide a new approach to releasing the charging difficulty in the analyses using electron beam as a probe, but also provide an insightful hint to the understanding of the charging processes in general.