Experimental variation in the spatial deposition of trace metals in feathers revealed using synchrotron X-ray fluorescence

Feathers can be used to investigate exposure to pollution in birds because they are a secondary route for the excretion of trace elements. Evidence based on analytical imaging and spectroscopy suggests that the spatial distribution of the essential trace element zinc within feathers is related to melanin pigmentation. However, our understanding of how trace elements are deposited into growing feathers is poor and has been hampered by a lack of analytical tools to examine the localization of trace elements within a feather. Here, synchrotron micro X-ray fluorescence spectroscopy was used to map zinc directly within the barb and barbules of lesser scaup (Aythya affinis) feathers grown after experimental increases in dietary zinc. The results showed distinct spatial variation in zinc within barbs and barbules, with higher levels observed in the latter. Furthermore, increases in dietary zinc were found to increase the relative levels of zinc throughout the barbules from the base to the tip of the feather. Finally, analysis of feather cross sections revealed that regions of the feather barb and barbules with higher melanosome density also contained higher levels of zinc. These results provide a more detailed understanding of zinc and melanosome arrangement within the feather barb and barbules. Moreover, these results provide further support for the use of feathers as a noninvasive tool to study exposure to trace elements and highlight the utility of X-ray spectroscopy in studies investigating impacts of a rapidly changing environment on wild bird health.     

Microspectroscopic soft X-ray analysis of keratin based biofibers

Scanning soft X-ray transmission microspectroscopy (STXM) and transmission electron microscopy (TEM) have been employed for a high-resolution morphological and chemical analysis of hair fibers from human, sheep and alpaca. STXM allows optimum contrast imaging of the main hair building blocks due to tuneable photon energy. Chemical similarities and deviations for the human hair building blocks as well as for the three investigated species are discussed on the basis of the local near-edge X-ray absorption fine structure (NEXAFS). The spectra of melanosomes corroborate the state-of-the-art model for the chemical structure of eumelanin. Complementary TEM micrographs reveal the occurrence of cortex sectioning in alpaca hair to some extent. A spectroscopic analysis for human hair cortex indicates low mass loss upon soft X-ray irradiation, but transformation of chemical species with decreasing amount of peptide bonds and increasing NEXAFS signal for unsaturated carbon–carbon bonds.     

Effects of dual-frequency slit ultrasound on the enzymolysis of high-concentration hydrolyzed feather meal: Biological activities and structural characteristics of hydrolysates

Ultrasound-assisted enzymolysis has been applied to improve conventional enzymolysis, while there are rare reports on the application of ultrasound to high-concentration feather protein enzymolysis. Therefore, the feasibility of dual-frequency slit ultrasound (DFSU) for enzymolysis of high-concentration hydrolyzed feather meal (HFM), as well as the biological activities and structural characteristics of hydrolysates were investigated. The single-factor test was used to optimize the ultrasonic processing parameters: substrate concentration, frequency mode, intermittent ratio, power density, and time. The results showed that protein recovery rate and conversion rate increased by 6.08% and 18.63% under the optimal conditions (200 g/L, 28/80 kHz, 5:2 s/s, 600 W/L, and 3 h) compared with conventional enzymolysis, respectively. The macromolecular proteins in hydrolysates were converted into micromolecular peptides (< 500 Da) when treated by DFSU, and antioxidant activity and angiotensin-I-converting enzyme (ACE) inhibitory activity of hydrolysates were increased. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) images illustrated the microstructure changes of feather protein particles in the ultrasound-assisted enzymatic hydrolysates of HFM (UEH), including more porous, smaller, and more uniform. Additionally, the conformation of protein molecules was significantly affected (P < 0.05), including the increase in free sulfhydryl (SH), the decrease in disulfide bond (SS) and surface hydrophobicity (H₀). Fourier transform infrared (FTIR) spectra analysis further showed that the secondary structure of feather proteins was modified with a reduction in α-helix, β-turn, and β-sheet, while an increase in random coil content was observed. These results indicated that DFSU could be a promising method to enhance high-concentration HFM for preparing peptide-rich hydrolysates with high antioxidant activity and ACE inhibitory activity.     

A closer look at the feather coloration in the male purple sunbird,Nectarinia asiatica

During the breeding season male, but not female, individuals of the purple sunbird possess colourful plumage of chiefly blue and black coloration with a splatter of orange and yellow on the chest. Representative feathers of these colours were collected from male birds during the breeding season and analyzed by reflectance and scanning electron microscopy. The rachis, which is the central support of a feather on which various barbs and barbules are arranged, is spongy and made up of keratin layers with rod-shaped melanosomes sparsely distributed within these layers. Barbs and barbules are the structural units of the feather and depending on how they are arranged provide a characteristic shape to the feather. X-Ray diffraction (XRD) analyses of the feathers revealed the presence of various metal elements that might contribute to the feathers’ colorations. Blue feathers are iridescent and contain mainly iron, copper, zinc and cobalt (in that order of abundance); black feathers were also found to contain mainly iron and copper, but chromium instead of zinc and cobalt, while yellow feathers were found to contain predominantly cobalt and nickel. The metal content of the feathers in an as yet unknown way may be involved in the production of the distinct absorbance and reflectance patterns that the brilliant plumage of the purple sunbird is renowned for.     

Backscattered electron imaging of micro- and nanostructures: 1. Method of analysis

A method for analysis of the formation of backscattered electron scanning electron microscope (SEM) images is described. The results of studying the formation of SEM signals obtained upon scanning an angular structure are given. The mechanisms of the formation of images in the modes of backscattered and slow secondary electrons are described.     

Scanning transmission X-ray microscopic analysis of purified melanosomes of the mouse iris

Melanosomes are specialized intracellular membrane bound organelles that produce and store melanin pigment. The composition of melanin and distribution of melanosomes determine the color of many mammalian tissues, including the hair, skin, and iris. However, the presence of melanosomes within a tissue carries potentially detrimental risks related to the cytotoxic indole–quinone intermediates produced during melanin synthesis. In order to study melanosomal molecules, including melanin and melanin-related intermediates, we have refined methods allowing spectromicroscopic analysis of purified melanosomes using scanning transmission X-ray microscopy. Here, we present for the first time absorption data for melanosomes at the carbon absorption edge ranging from 284 to 290 eV. High-resolution images of melanosomes at discrete energies demonstrate that fully melanized mature melanosomes are internally non-homogeneous, suggesting the presence of an organized internal sub-structure. Spectra of purified melanosomes are complex, partially described by a predominating absorption band at 288.4 eV with additional contributions from several minor bands. Differences in these spectra were detectable between samples from two strains of inbred mice known to harbor genetically determined melanosomal differences, DBA/2J and C57BL/6J, and are likely to represent signatures arising from biologically relevant and tractable phenomena.     

Colonization of cashew plants by Lasiodiplodia theobromae: microscopical features

Lasiodiplodia theobromae is a phytopathogenic fungus causing gummosis, a threatening disease for cashew plants in Brazil. In an attempt to investigate the ultrastructural features of the pathogen colonization and its response to immunofluorescence labeling, light, confocal and electron microscope studies were conducted on different severity scale patterns of diseased plants. Lasiodiplodia-antisera was checked for cross reactivity against common cashew plants fungi. Optical microscopy analysis revealed a longitudinally sectioned hyphae located within the xylem vessels, showing an extensive hyphal development in the secondary xylem tissue. SEM images demonstrated that the fungus was found in some asymptomatic samples, particularly within the xylem vessels as confirmed by the optical images. Symptomatic sample images showed an extensive distribution of the fungus along the secondary xylem, within the vessels, infecting xylem parenchyma. A closer look in the secondary xylem parenchyma reveals a heavy and profuse invasion of the cells with a distinguishable cell wall disintegration and fully hyphae dispersal. There was no reactivity of Lasiodiplodia-antisera against mycelial extracts of Colletotrichum gloeosporioides, Phomopsis anardii and Pestalotiopsis guepinii. Following incubation of sections with the polyclonal antisera, the hyphae were intensely and regularly labeled. Rays, vessels and parenchyma cells were the preferred pathway for L. theobromae colonization. Artificial infection provides the information that the vascular cylinder is undoubtedly employed and used by the fungus for hyphae distribution. Immunofluorescence assay employed in situ was applied and the polyclonal antisera produced was able to recognize the fungus and proved to be a sensitive technique to detect it.     

Monitoring the effect of sonoporation on the cells using electrochemical approach

Sonoporation is applied to enhance the permeability of the cell to bioactive materials by employing the acoustic cavitation of microbubbles. This phenomena would be helpful in molecular biology, delivery of large molecules into the cells and gene therapy. Many methods have been applied to monitor the biological effects and trace of sonoporation on the cells such as scanning/transmission electron microscopy, confocal imaging and flow cytometry. Here, we monitored the effect of sonoporation on the cells using electrochemical method with an integrated three electrode system. Electrochemical responses of stimulated cells, compared to flow cytometry and electron microscopy results, presented different patterns of sonoporation in the cells detectable by cyclic voltammetry. In addition, confocal microscopy from actin stress fibers and young’s modulus measured by AFM revealed the correlation of cell mechanics and amount of induced sonopores in the cells. This method could be applied as a new trend in cellular mechanochemical studies.     

体视学与激光共聚焦显微镜图像分析黑色素瘤细胞三维形态学参数的变化

体视学是由形态学与数学交叉形成的一门新兴学科,通过二维结构信息定量测量和分析三维形态结构特征。采用体视学点计数方法,对肿瘤细胞B16F10、B16/Vector及B16/GPR4各20组的共聚焦显微镜图像进行测量,定量分析了其三维形态参数的差异。对比三类细胞的体视学参数发现,对照组B16/Vector和原始细胞B16F10没有明显差异,实验组B16/GPR4的细胞比表面及相对形状因子参数与另两类细胞相比均表现出了显著性差异(p值分别小于0.05和0.02,即B16/GPR4细胞形态光滑,没有较多的突触和伪足),与前期Transwell小室细胞侵袭及迁移实验研究结果相吻合,体现出B16/GPR4细胞中G蛋白耦联受体4(GPR4)基因对细胞迁移性抑制的生物特性。研究结果表明,体视学和激光扫描共聚焦显微镜细胞图像的结合,可以实现细胞三维结构参数的快速测量,是细胞生物学尤其是细胞形态学分析的有效研究方法。     

Mechanism of electron multiplication due to charging for a SiO2 sample with a buried microstructure in SEM: A simulation analysis

This study investigates the mechanism of electron redistribution and multiplication for a SiO₂ sample with a buried structure in scanning electron microscopy by numerical simulation. The simulation involved electron scattering and internal charge transport in the sample, the tracking of emitted secondary electrons (SEs), and the generation of tertiary electrons (TEs) produced by returned SEs due to charging of the sample. The results show that a buried grounded structure causes a non-uniform distribution of surface potential, and an electric field above the surface. As a result, although the number of escaped SEs above the margin of the buried structure decreases, the number of generated TEs increases more, leading to a final current of electrons that include escaped SEs and increased TEs. This multiplication of SEs might make a crucial contribution to the abnormal negative-charging contrast in SEM. During the electron beam irradiation, the variation in the number of total escaped electrons presents an obvious increase after an initial slight decrease, which corresponded to the transient characteristics of gray levels in SEM images from dark to abnormally bright.     

Hypokinetic stress and neuronal porosome complex in the rat brain: the electron microscopic study

Porosomes are the universal secretory machinery in cells, where membrane-bound secretory vesicles transiently dock and fuse to release intravesicular contents to the outside of the cell during cell secretion. Studies using atomic force microscopy, electron microscopy, electron density and 3D contour mapping, provided rich nanoscale information on the structure and assembly of proteins within the neuronal porosome complex in normal brain. However it remains uncertain whether pathological conditions that alter process of neurotransmission, provoke alterations in the porosome structure also. To determine if porosomes are altered in disease states, the current study was undertaken for first time using high resolution electron microscope. One of pathologies that produce subtle alteration at the presynaptic terminals has been demonstrated to be hypokinetic stress. The central nucleus of amygdale is the brain region, where such alterations are mostly expressed. We have examined the width and depth of the neuronal porosome complex and their alterations provoked by chronic hypokinetic stress in above mentioned limbic region. Specifically, we have demonstrated that despite alterations in the presynaptic terminals and synaptic transmission provoked by this pathological condition in this region, the final step/structure in neurosecretion--the porosome--remains unaffected: the morphometric analysis of the depth and diameter of this cup-shaped structure at the presynaptic membrane point out to the heterogeneity of porosome dimensions, but with unchanged fluctuation in norm and pathology.     

Backscattered electron imaging for reduced charging of moisturized corn starch granules: implications for versatile imagery of hygroscopic powder specimens

Charging artifacts and surface features of corn starch granules were investigated by scanning electron microscopy. Three types of industrial waxy corn starch granules with different levels of moisture content (0, 10.3, and 24.2%) were prepared and subjected to both secondary electron imaging and backscattered electron imaging. There were no significant charging artifacts in secondary electron images at 3 or 5 kV. However, imaging at higher magnifications and accelerating voltages much lower than 3 kV ranging from 0.1 to 1 kV did not show well-resolved structures. At higher accelerating voltages than 5 kV, charging was manifested as excessive brightness at specific areas and alteration of bright and dark lines in the direction of the raster pattern in secondary electron images of all the types of specimens tested. As the accelerating voltage increased up to 30 kV in secondary electron images, the charging also increased. Meanwhile, no charging was detected in all the backscattered electron images taken at different accelerating voltages. As the accelerating voltage increased in backscattered electron images, the resolution increased with less depth of focus. Consistent results were found in all the types of corn starch granules assayed in this study. These results suggest that a simple and rapid morphological analysis of moisturized starches can be performed by backscattered electron imaging without considerable heat drying of starches. Concomitantly, it allows for imposing a higher accelerating voltage to ensure better image resolution, facilitating morphological characterization of diverse starch granules as they are in native states.     

Methods of calculating the band structure and low-energy secondary electron spectroscopy of iridium

A theoretical interpretation is put forward for the fine structure of the secondary electron emission spectra of Ir normal to the (111) surface and the total current spectrum of an Ir polycrystal. The calculations took into account the energy dependence of the broadening of the energy band levels, the electron-electron and electron-plasmon contributions to the nonequilibrium electron distribution function, and the isotropic component of the current from the electrons scattered at the surface. It is shown that the fine structure of the secondary electron emission spectrum and the total current spectrum is mainly attributable to the electron structure of the final states into which the electrons enter or from which they are emitted so that the characteristics of the band configuration in the energy band structure can be reconstructed directly from the experimental data. This method can be used to separate bulk effects from surface effects in the secondary electron emission and total current spectra. It is confirmed that the fine structure of the secondary electron emission and total current spectra depends on the geometric structure and the degree of ordering of the crystals. A reduction in the intensity of the fine structure serves as a measure of the defect structure in the surface region of the sample which can be successfully used to monitor the surface state during treatment. Zh. Tekh. Fiz. 69, 94–96 (June 1999)     

One-step hydrothermal synthesis of feather duster-like NiS@MoS<Subscript>2</Subscript> with hierarchical array structure for the Pt-free dye-sensitized solar cell

Novel feather duster-like nickel sulfide (NiS) @ molybdenum sulfide (MoS₂) with hierarchical array structure is synthesized via a simple one-step hydrothermal method, in which a major structure of rod-like NiS in the center and a secondary structure of MoS₂ nanosheets with a thickness of about 15–55 nm on the surface. The feather duster-like NiS@MoS₂ is employed as the counter electrode (CE) material for the dye-sensitized solar cell (DSSC), which exhibits superior electrocatalytic activity due to its feather duster-like hierarchical array structure can not only support the fast electron transfer and electrolyte diffusion channels, but also can provide high specific surface area (238.19 m² g^?1) with abundant active catalytic sites and large electron injection efficiency from CE to electrolyte. The DSSC based on the NiS@MoS₂ CE achieves a competitive photoelectric conversion efficiency of 8.58%, which is higher than that of the NiS (7.13%), MoS₂ (7.33%), and Pt (8.16%) CEs under the same conditions.

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Graphical abstract Novel feather duster-like NiS@MoS₂ hierarchical structure array with superior electrocatalytic activity was fabricated by a simple one-step hydrothermal method.

     

Compositional contrast of uncoated fungal spores and stained section-face by low-loss backscattered electron imaging

Comparative surface imaging was performed on uncoated fungal spores and stained section-face by field emission scanning electron microscopy with an in-column energy-selective backscattered electron detector. Epoxy resin thin sections (ca. 200 and 500 nm thick) of the osmicated and uranyl acetate/lead citrate-stained fungus were examined with the microscope. Topographical contrast was evident in secondary electron imaging by either a below-lens or an in-lens detector. Meanwhile, low-loss backscattered electron images showed mainly compositional contrast at low accelerating voltages (mostly below 1 kV). With attenuated topographical contrast, several different electron densities could be detected, exhibiting several levels of electron density even on a flat plane of spines. Minute differences in topography on epoxy resin sections as seen by secondary electron imaging represented the periphery of the fungal spores and hyphae. On the other hand, the compositional contrast could be retrieved from stained section-face in low-loss BSE imaging, revealing subcellular entities after contrast inversion. The resolution of low-loss BSE imaging was sufficient to resolve plasma membrane, and various types of vacuoles and vesicles. These results suggest that low-loss backscattered electron imaging could potentially provide compositional information to resolve surface chemical features of uncoated microbial cells and stained section-face with heterogeneous surface compositions.     

Ultrastructural description of spermiogenesis within the Mediterranean Gecko, Hemidactylus turcicus (Squamata: Gekkonidae)

We studied spermiogenesis in the Mediterranean Gecko, Hemidactylus turcicus, at the electron microscope level and compared to what is known within other Lepidosaurs. In H. turcicus germ cells are connected via cytoplasmic bridges where organelle and cytoplasm sharing is observed. The acrosome develops from merging transport vesicles that arise from the Golgi and subsequently partition into an acrosomal cap containing an acrosomal cortex, acrosomal medulla, perforatorium, and subacrosomal cone. Condensation of DNA occurs in a spiral fashion and elongation is aided by microtubules of the manchette. A nuclear rostrum extends into the subacrosomal cone and is capped by an epinuclear lucent zone. Mitochondria and rough endoplasmic reticulum migrate to the posterior portion of the developing germ cell during the cytoplasmic shift and the flagellum elongates. Mitochondria surround the midpiece as the anlage of the annulus forms. The fibrous sheath begins at mitochondrial tier 3 and continues into the principal piece. Peripheral fibers associated with microtubule doublets 3 and 8 are grossly enlarged. During the final stages of germ cell development spermatids are wrapped with a series of Sertoli cell processes, which exhibit ectoplasmic specializations and differing cytoplasmic consistencies. The results observed here corroborate previous studies, which show the conservative nature of sperm morphology. However, ultrastructural character combinations specific to sperm and spermiogenesis seem to differ among taxa. Further studies into sperm morphology are needed in order to judge the relevance of the ontogenic changes recorded here and to determine their role in future studies on amniote evolution.     

Analysis of the healthy rabbit lens surface using MAC Mode atomic force microscopy

In this investigation healthy rabbit crystalline lenses were characterized by atomic force microscopy (AFM). The lenses were cut in slices with thickness with 1mm and thus, put after cortex distinct regions of nucleus and cortex for AFM examination. AFM analysis were carried out using a PicoSPM I operating in Mac Mode. We obtained topographic images of rabbit lenses and a quantitative analysis of the width and height of fibers for nucleus and cortex regions. The longitudinal section analysis of fibers in the nucleus region indicated structures with an average width of 200nm and average height of 200nm. The intershells distance was determined as 4microm. Fiber cell cross-section dimensions, longitudinal and transverse widths, could be estimated in these regions from the AFM images. Structures with average widths as small as 1.0microm are observed in the nucleus; the intershell distance is 4.0microm. In cortical regions, hexagonal structures with average longitudinal and transverse widths of 5.0mum and 3.0mum, respectively, were identified. Three-dimensional images of tissue sections with resolution on a nanometer scale were obtained. The potential of AFM analysis for characterizing healthy and pathologic lens tissues is discussed.     

Development of computer-assisted minimal-dose system with beam blanker for TEM

A computer-assisted minimal-dose system has been developed for the high-resolution observation of radiation-sensitive samples using a transmission electron microscope (TEM). This system consists of a CCD camera, a beam blanker and a control computer (PC) that also controls the TEM. A sample is illuminated by an electron beam only when the CCD camera takes images; otherwise, the beam blanker cuts off the electron beam. Emulated images, which are calculated from the images taken and the variable parameters of the TEM, such as magnification and sample stage position, are displayed on the control PC display. After a few times of repetition of exposures and emulations, the sample is positioned to final observation area. Subsequently to select the observation area, the system automatically adjusts the focus position from two different illuminating angle images and the TEM conditions are appropriate for taking a final image. The total electron dose before the final image is taken can be markedly decreased because the sample is irradiated by an electron beam only when images are taken. For the final image, a three-dimensional Fourier filtering method (3DFFM), which corrects the spherical aberration for the through-focus image series and enables the selection of the optimum focus condition later, is also included in our system.     

Chromatoid body: remnants of nucleolar proteins during spermatogenesis in triatomine (Heteroptera, Triatominae)

In this study, we analyzed fibrillarin nucleolar protein expression in CBs of spermatogenic cells from testicular follicles of Triatoma sordida and Triatoma infestans. In the structural and ultrastructural analysis, it was used impregnation by silver ions, immunocytochemistry, immunofluorescence and transmission electron microscopy using antibodies against fibrillarin. Regarding the results, the fibrillarin nucleolar protein marked the nucleus and some cytoplasmic spots of germ cells during spermatogenesis in triatomines. These data suggest that fibrillarin could be a constituent of the CB that was most likely derived from nucleolar fragmentation. This is the first time that fibrillarin protein expression has been shown in the CB during spermatogenesis progression in triatomines. The knowledge regarding CB constituents may help to expand the understanding of the physiological role of this structure and the role that it plays in the reproductive biology of triatomines, which are vectors of Chagas Disease.