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Adjuvants are substances that enhance adaptive immune responses when formulated in a vaccine. Alum and MF59 are two vaccine adjuvants licensed for human vaccination. Their mode of action has not been completely elucidated. Here we show the first ultrastructural visualization of Alum and MF59 interaction with immune cells in vitro and in vivo. We observed that Alum is engulfed by cells as inclusions of laminae that are detectable within draining lymph nodes. MF59 is instead engulfed by cells in vitro as low-electron-dense lipid-like inclusions that display a vesicle pattern, as confirmed by confocal microscopy using fluorescently labeled MF59. However, lipid-like inclusions with different high- and low-electron-dense content are detected within cells of draining lymph nodes when injecting MF59. As high-electron-dense lipid-like inclusions are also detected upon injection of Alum, our results suggest that the low-electron-dense inclusions are formed by engulfed MF59, whereas the high-electron-dense inclusions are proper lipid inclusions. Thus, we demonstrated that vaccine adjuvants are engulfed as inclusions by lymph node cells and hypothesize that adjuvant treatment may modify lipid metabolism.
The distribution of boron in tissue samples coming from boron neutron capture therapy protocols can be determined through the analysis of its autoradiography image on a nuclear track detector. A more precise knowledge of boron atom location on the microscopic scale can be attained by the observation of nuclear tracks superimposed on the sample image on the detector. A method to produce an “imprint” of cells cultivated on a polycarbonate detector was developed, based on the photodegradation properties of UV-C radiation on this material. Optimal conditions to generate an appropriate monolayer of Mel-J cells incubated with boronophenylalanine were found. The best images of both cells and nuclear tracks were obtained for a neutron fluence of 1013 cm−2, 6 h UV-C (254 nm) exposure, and 4 min etching time with a KOH solution. The imprint morphology was analyzed by both light and scanning electron microscopy. Similar samples, exposed to UV-A (360 nm) revealed no cellular imprinting. Etch pits were present only inside the cell imprints, indicating a preferential boron uptake (about threefold the incubation concentration). Comparative studies of boron absorption in different cell lines and in vitro evaluation of the effect of diverse boron compounds are feasible with this methodology.
When bone is exposed to the burial environment it may experience structural changes induced by multiple agents. The study of postmortem alterations is important to differentiate decomposition phenomena from normal physiological processes or pathological lesions, as well as to assess bone tissue quality. Microscopy is of great utility to evaluate the integrity of bone microstructure and it provides significant data on long-term bone decomposition. A total of 18 human bone sections (eight archeological and ten retrieved from an identified skeletal collection) were selected for analysis under plane light and polarized light. The aim of this exploratory study was to analyze the impact of diagenesis and taphonomy on the bone microstructure, as well as on the differential diagnosis of pathological conditions. The results showed that the microscopy approach to bone tissues contributed materially as an aid in the detailed description of the main diagenetic changes observed. It showed that gross inspection does not provide a realistic assessment of bone tissue preservation, which can impact in the characterization of lesions present and subsequent disease diagnosis. Therefore, researchers should continue to consider the application of histological techniques if the aim is to comprehend tissue integrity and its association with decomposition or disease.
We here present two new methods for the characterization of fluorescent localization microscopy images obtained from immunostained brain tissue sections. Direct stochastic optical reconstruction microscopy images of 5-HT1A serotonin receptors and glial fibrillary acidic proteins in healthy cryopreserved brain tissues are analyzed. In detail, we here present two image processing methods for characterizing differences in receptor distribution on glial cells and their distribution on neural cells: One variant relies on skeleton extraction and adaptive thresholding, the other on k-means based discrete layer segmentation. Experimental results show that both methods can be applied for distinguishing classes of images with respect to serotonin receptor distribution. Quantification of nanoscopic changes in relative protein expression on particular cell types can be used to analyze degeneration in tissues caused by diseases or medical treatment.
The aim of the present research was to trace CD34+ stromal fibroblastic cells (CD34+ SFCs) in the palatal connective tissue harvested for muco-gingival surgical procedures and in granulation tissues from periodontal pockets using immunohistochemical and transmission electron microscopy. Immunohistochemical analysis targeted the presence of three antigens: CD31, α-smooth muscle actin (α-SMA), and CD34. In the palate, CD31 staining revealed a colored inner ring of the vessels representing the endothelium, α-SMA+ was located in the medial layer of the vasculature, and CD34 was intensely expressed by endothelial cells and artery adventitial cells (considered to be CD34+ SFCs). Granulation tissue showed the same pattern for CD31+ and α-SMA, but a different staining pattern for CD34. Ultrastructural examination of the palatal tissue highlighted perivascular cells with fibroblast-like characteristics and pericytes in close spatial relationship to endothelial cells. The ultrastructural evaluation of granulation tissue sections confirmed the presence of neovasculature and the inflammatory nature of this tissue. The present study traced the presence of CD34+ SFCs and of pericytes in the palatal connective tissue thus highlighting once more its intrinsic regenerative capabilities. The clinical and systemic factors triggering mobilization and influencing the fate of local CD34+SCFs and other progenitors are issues to be further investigated.
This study evaluated the influence of tubular density of different dentin depths and location on the bond strength of high-viscosity glass ionomer cements (GIC). A total of 20 molars were selected and assigned into six experimental groups, considering two different high-viscosity GICs—Fuji IX (FIX) or Ketac Molar (KM), and dentin location—proximal, occlusal superficial, or occlusal deep dentin (n=10). Teeth were cut and a topographical analysis of four sections per group was performed to obtain data about the tubular density of each different dentin location and depths by laser scanning confocal microscopy (100×). Polyethylene tubes were placed over the pretreated surfaces and filled with one of the GICs. Microshear bond strength (µSBS) test was performed after storage in distilled water (24 h at 37°C). Failure modes were evaluated using a stereomicroscope (400×). Multilevel regression analysis was performed to compare the results at a significance level set at 5%. The tubule density was inversely proportional to the bond strength for both GICs (p<0.05). Adhesive/mixed failure prevailed in all experimental groups. Proximal (30036.5±3433.3) and occlusal superficial 29665.3±1434.04 dentin shows lower tubule density, resulting in a better GIC bonding performance (proximal: FIX–3.61±1.05; KM–3.40±1.62; occlusal superficial: FIX–4.70±1.85; KM–4.97±1.25). Thus, we can concluded that the lowest tubule density in proximal and occlusal superficial dentin results in a better GIC bond strength performance.
This work developed a phase congruency algorithm combined with texture analysis to quantitatively characterize collagen morphology in second-harmonic generation (SHG) images from human scars. The extracted phase and texture parameters of the SHG images quantified collagen directionality, homogeneity, and coarseness in scars and varied with scar duration. Phase parameters showed an increasing tendency of the mean of phase congruency with scar duration, indicating that collagen fibers are better oriented over time. Texture parameters calculated from local difference local binary pattern (LD-LBP) and Haar wavelet transform, demonstrated that the LD-LBP variance decreased and the energy of all subimages increased with scar duration. It implied that collagen has a more regular pattern and becomes coarser with scar duration. In addition, the random forest regression was used to predict scar duration, demonstrating reliable performance of the extracted phase and texture parameters in characterizing collagen morphology in scar SHG images. Results indicate that the extracted parameters using the proposed method can be used as quantitative indicators to monitor scar progression with time and can help understand the mechanism of scar progression.
Mechanical properties of the arterial wall depend largely on orientation and density of collagen fiber bundles. Several methods have been developed for observation of collagen orientation and density; the most frequently applied collagen-specific manual approach is based on polarized light (PL). However, it is very time consuming and the results are operator dependent. We have proposed a new automated method for evaluation of collagen fiber direction from two-dimensional polarized light microscopy images (2D PLM). The algorithm has been verified against artificial images and validated against manual measurements. Finally the collagen content has been estimated. The proposed algorithm was capable of estimating orientation of some 35 k points in 15 min when applied to aortic tissue and over 500 k points in 35 min for Achilles tendon. The average angular disagreement between each operator and the algorithm was −9.3±8.6° and −3.8±8.6° in the case of aortic tissue and −1.6±6.4° and 2.6±7.8° for Achilles tendon. Estimated mean collagen content was 30.3±5.8% and 94.3±2.7% for aortic media and Achilles tendon, respectively. The proposed automated approach is operator independent and several orders faster than manual measurements and therefore has the potential to replace manual measurements of collagen orientation via PLM.
Single-particle reconstruction (SPR) and electron crystallography (EC), two major applications in electron microscopy, can be used to determine the structure of membrane proteins. The three-dimensional (3D) map is obtained from separated particles in conventional SPR, but from periodic unit cells in EC. Here, we report a refined SPR procedure for processing 2D crystal images. The method is applied to 2D crystals of melibiose permease, a secondary transporter in Escherichia coli. The current procedure is improved from our previously published one in several aspects. The “gold standard Fourier shell correlation” resolution of our final reconstruction reaches 13 Å, which is significantly better than the previously obtained 17 Å resolution. The choices of different refinement parameters for reconstruction are discussed. Our refined SPR procedure could be applied to determine the structure of other membrane proteins in small or locally distorted 2D crystals, which are not ideal for EC.
In biology, hemocytometers such as Malassez slides are widely used and are effective tools for counting cells manually. In a previous work, a robust algorithm was developed for grid extraction in Malassez slide images. This algorithm was evaluated on a set of 135 images and grids were accurately detected in most cases, but there remained failures for the most difficult images. In this work, we present an optimization of this algorithm that allows for 100% grid detection and a 25% improvement in grid positioning accuracy. These improvements make the algorithm fully reliable for grid detection. This optimization also allows complete erasing of the grid without altering the cells, which eases their segmentation.
Stereological techniques using the optical disectors require estimation of final section thickness, but frozen tissue irregularities may interfere with this estimation. Cryostat slices from rodent nerve tissues (dorsal root ganglia, spinal cord, and brain), cut at 16, 40, and 50 μm, were digitized with a confocal microscope and visualized through 3D software. Geometric section thickness of tissue (Tgeom) was defined as tissue volume/area. Maximal section thicknesses (Tmax), from the top to the bottom of the section, were measured in a random sample of vertical ZX planes. Irregularities were mostly related to blood vessels traversing the tissue and neuronal somas protruding over the cut surfaces, with other neuron profiles showing a fragmented appearance. Irregularities contributed to increasing the distance between the tops and bottoms of slices sectioned in different laboratories. Significant differences were found between Tmax and Tgeom for all thickness studies and counting frames (p<0.01). The Tgeom/Tmax average rate was 68.4–85.7% in volumes around cell profiles (∼600–1,200 μm2) and 83.3–91.8% in subcellular samples (∼25–160 μm2). Confocal microscopy may help to assess tissue irregularities, which might lead to an overestimation of tissue volume if section thickness is estimated by focusing on the top and bottom of the sections.
Although the advent of microwave technologies has both improved and accelerated tissue processing for microscopy, there still remain many limitations in conventional chemical fixation, dehydration, embedding, and sectioning, particularly with regard to plant materials. The Proteaceae, a family of plants widely distributed in the Southern Hemisphere and well adapted to harsh climates and nutrient-poor soils, is a perfect example; the complexity of Proteaceae leaves means that almost no ultrastructural data are available as these are notoriously difficult to both infiltrate and section. Here, a step-by-step protocol is described that allows for the successful preparation of Banksia prionotes (Australian Proteaceae) leaves for both light and transmission electron microscopy. The method, which applies a novel combination of vibratome sectioning, microwave processing and vacuum steps, and the utilization of an ultra low viscosity resin, results in highly reproducible, well-preserved, sectionable material from which very high-quality light and electron micrographs can be obtained. With this, cellular ultrastructure from the level of a leaf through to organelle substructure can be studied. This approach will be widely applicable, both within and outside of the plant sciences, and can be readily adapted to meet specific sample requirements and imaging needs.
This paper presents experimental results obtained from a scanning electron microscope (SEM) second-order focusing toroidal electron energy analyzer attachment. The results demonstrate that the analyzer can be used to obtain high signal-to-noise voltage and dopant concentration measurements on semiconductors in the presence of different electric field conditions at the sample. The experimentally calculated relative error of measurement typically varies from 31 to 63, corresponding to secondary electron (SE) signal mean shifts of 9–18 mV. The millivolt accuracy of these results is over one order of magnitude better than earlier quantitative dopant concentration measurements made by a retarding field analyzer.
Austenitic 316L stainless steel can be used for orthopedic implants due to its biocompatibility and high corrosion resistance. Its range of applications in this field could be broadened by improving its wear and friction properties. Surface properties can be modified through surface hardening treatments. The effects of such treatments on the microstructure of the alloy were investigated here. Surface Mechanical Attrition Treatment (SMAT) is a surface treatment that enhances mechanical properties of the material surface by creating a thin nanocrystalline layer. After SMAT, some specimens underwent a plasma nitriding process to further enhance their surface properties. Using electron backscatter diffraction, transmission Kikuchi diffraction, energy dispersive spectroscopy, and transmission electron microscopy, the microstructural evolution of the stainless steel after these different surface treatments was characterized. Microstructural features investigated include thickness of the nanocrystalline layer, size of the grains within the nanocrystalline layer, and depth of diffusion of nitrogen atoms within the material.
The performance of polycrystalline CdTe photovoltaic thin films is expected to depend on the grain boundary density and corresponding grain size of the film microstructure. However, the electrical performance of grain boundaries within these films is not well understood, and can be beneficial, harmful, or neutral in terms of film performance. Electron backscatter diffraction has been used to characterize the grain size, grain boundary structure, and crystallographic texture of sputtered CdTe at varying deposition pressures before and after CdCl2 treatment in order to correlate performance with microstructure. Weak fiber textures were observed in the as-deposited films, with (111) textures present at lower deposition pressures and (110) textures observed at higher deposition pressures. The CdCl2-treated samples exhibited significant grain recrystallization with a high fraction of twin boundaries. Good correlation of solar cell efficiency was observed with twin-corrected grain size while poor correlation was found if the twin boundaries were considered as grain boundaries in the grain size determination. This implies that the twin boundaries are neutral with respect to recombination and carrier transport.
Understanding the morphological structure of membranes is essential to improve performance of membrane-based applications. In this paper, macroporous membranes were investigated and two methods introduced as an alternative for characterization of stereo-structure of the membranes. We combined the use of synchrotron X-ray nanotomography and small-angle X-ray scattering to examine the internal structure of cellulose acetate membranes with studies of the capture of surface-modified gold nanoparticles within these membranes. Finally, the morphological structures of macroporous membranes were visualized and their relationships with penetration tendency of surface-modified gold nanoparticles were explained.
Here, we report reproducible and accurate measurement of crystallographic parameters using scanning transmission electron microscopy. This is made possible by removing drift and residual scan distortion. We demonstrate real-space lattice parameter measurements with <0.1% error for complex-layered chalcogenides Bi2Te3, Bi2Se3, and a Bi2Te2.7Se0.3 nanostructured alloy. Pairing the technique with atomic resolution spectroscopy, we connect local structure with chemistry and bonding. Combining these results with density functional theory, we show that the incorporation of Se into Bi2Te3 causes charge redistribution that anomalously increases the van der Waals gap between building blocks of the layered structure. The results show that atomic resolution imaging with electrons can accurately and robustly quantify crystallography at the nanoscale.
Microstructure evolution during the formation of B2–NiAl by high energy ball milling of equiatomic elemental mixtures was studied by X-ray diffractometer, scanning electron microscopy, and transmission electron microscopy (TEM). The crystallite size, lattice defects and ordering of the B2–NiAl were monitored via TEM as function of milling time. The diffusion reaction, Ni+Al→NiAl3 or/and Ni2Al3, occurred during high energy ball milling, and to a certain extent offered the stored energy for the explosive exothermic reaction, Ni+Al→B2–NiAl. The fine microstructure of newly formed B2–NiAl after 5 h milling involved high density defects, e.g. antiphase boundary, long range ordering domains, vacancies, and dislocations.
Data-constrained modeling is a method that enables three-dimensional distribution of mineral phases and porosity in a sample to be modeled based on micro-computed tomography scans acquired at different X-ray energies. Here we describe an alternative method for measuring porosity, synchrotron K-edge subtraction using xenon gas as a contrast agent. Results from both methods applied to the same Darai limestone sample are compared. Reasonable agreement between the two methods and with other porosity measurements is obtained. The possibility of a combination of data-constrained modeling and K-edge subtraction methods for more accurate sample characterization is discussed.
Using an in situ load frame within a scanning electron microscope, a microstructural section on the surface of an annealed tantalum (Ta) polycrystalline specimen was mapped at successive tensile strain intervals, up to ~20% strain, using electron backscatter diffraction. A grain identification and correlation technique was developed for characterizing the evolving microstructure during loading. Presenting the correlated results builds on the reference orientation deviation (ROD) map concept where individual orientation measurements within a grain are compared with a reference orientation associated with that grain. In this case, individual orientation measurements in a deformed grain are measured relative to a reference orientation derived from the undeformed (initial) configuration rather than the current deformed configuration as has been done for previous ROD schemes. Using this technique helps reveal the evolution of crystallographic orientation gradients and development of deformation-induced substructure within grains. Although overall crystallographic texture evolved slowly during deformation, orientation spread within grains developed quickly. In some locations, misorientation relative to the original orientation of a grain exceeded 20° by 15% strain. The largest orientation changes often appeared near grain boundaries suggesting that these regions were preferred locations for the initial development of subgrains.