Electron energy loss spectroscopy for analysis of inhaled ultrafine particles in rat lungs

Epidemiologic studies have associated cardiovascular morbidity and mortality with ambient particulate air pollution. Particles smaller than 100 nm in diameter (ultrafine particles) are present in the urban atmosphere in very high numbers yet at very low mass concentration. Organs beyond the lungs are considered as targets for inhaled ultrafine particles, whereby the route of particle translocation deeper into the lungs is unclear. Five rats were exposed to aerosols of ultrafine titanium dioxide particles of a count median diameter of 22 nm (geometric standard deviation, GSD 1.7) for 1 hour. The lungs were fixed by intravascular perfusion of fixatives immediately thereafter. TiO(2) particles in probes of the aerosol as well as in systematic tissue samples were analyzed with a LEO 912 transmission electron microscope equipped with an energy filter for elemental microanalysis. The characteristic energy loss spectra were obtained by fast spectrum acquisition. Aerosol particles as well as those in the lung tissue were unambiguously identified by electron energy loss spectroscopy. Particles were mainly found as small clusters with a rounded shape. Seven percent of the particles in the lung tissue had a needle-like shape. The size distribution of the cluster profiles in the tissue had a count median diameter of 29 nm (GSD 1.7), which indicates no severe clustering or reshaping of the originally inhaled particles. Electron energy loss spectroscopy and related analytical methods were found to be suitable to identify and localize ultrafine titanium dioxide particles within chemically fixed and resin-embedded lung tissue.     

Morphological aspects of particle uptake by lung phagocytes

Macrophages residing on the inner epithelial surfaces of airways and alveoli are the only lung phagocytes exposed directly to the environment. Their phagocytic and microbicidal activities are essential for maintaining this organ in a clean and sterile state. The morphology of these phagocytes can be investigated in situ only after implementing special techniques, which involve intravascular triple-perfusion of aqueous fixatives or instillation of nonpolar ones. Such studies have revealed the engulfment of particles by these cells to be rapid, the process being essentially complete within a day. Particles are entrapped within phagosomes and the host cells eventually transported out of the lungs by mucociliary action, macrophages with higher loads being more rapidly eliminated than those with lower ones. Very small particles or those persisting on the epithelial surfaces may be taken up by the eponymous cells. Translocation of particles into the underlying connective tissue and their subsequent phagocytosis by interstitial macrophages prolongs their retention time in the lungs. The still poorly studied pleural macrophages might be involved in cell-mediated immune responses within the pleural space. Intravascular pulmonary macrophages figure largely in the phagocytosis of circulating particles. The role played by dendritic cells in particle uptake by the lungs is not well understood. Airway and alveolar macrophages are the primary phagocytes of the lung. In nonoverload situations and for particles >1 microm, a small proportion of those recruited suffices to remove material from the epithelial surface before other phagocytes, with an apparently greater immunological potential, gain access to it.     

Peribronchiolar accumulation of dendritic cells and their close association with CD4(+) T cells in the murine lung hypersensitivity

In order to understand the interaction between dendritic cells (DCs) and helper T (Th) cells in the region exposed to antigens during pulmonary delayed-type hypersensitivity (DTH), which is considered to be mediated by Th1 cells, we immunohistochemically investigated their spatial relationship in the cellular infiltrate. At 24 hours after intratracheal instillation of hapten in sensitized mice, DCs were preferentially accumulated around the bronchioles, whereas macrophages were more abundant around the accompanying arteries. DCs often formed a cluster, in which they were interconnected with each other by projections. Serial section analysis revealed that clustered DCs made a close apposition to Th cells but much less frequently to cytotoxic T cells and B cells. Immunoelectron microscopy demonstrated that lymphocytes extravasated the capillaries in the peribronchiolar interstitium and made conjugation with DCs. In the interstitial tissue, DCs often adhered to the fibroblasts, suggesting the supportive role of the latter cells in DC migration. Eosinophils were also frequent around the arteries, representing the possible involvement of Th2 cytokines. By contrast, in a chronic type of airway inflammation induced by repeated challenges of aerosolized ovalbumin, DCs were densely and diffusely accumulated around the arteries in the same way as macrophages. The present study demonstrated a close association of DCs with Th cells around the bronchioles during pulmonary DTH, suggesting that local interaction between them in the lung may play important roles in the development of this disorder.     

Section staining for electron microscopy using tannic acid as a mordant: a simple method for visualization of glycogen and collagen.

The possibility that tannic acid and other classical mordants can be used in the electron microscopical section staining technique has been tested. A mordant can be defined as a chemical that combines with both a certain specific tissue component and the staining substance and thereby permits a staining reaction that otherwise will not be obtained. The following features were found to characterize section staining of tannic acid mordanted sections. Tannic acid apparently blocks those sites that normally would be contrasted by uranyl acetate or some other staining compounds. Ribosomes remain unstained. Glycogen particles, on the other hand, were stained, whereas they are not in non-mordanted sections. In fact, glycogen was the only cytoplasmic component to be contrasted by the uranyl acetate, and collagen the only extracellular component. Several different section staining solutions gave the same staining patterns of examined cells and tissues. Specificity of the reaction thus seems to depend on the mordant rather than on the heavy atom section stain. Some other tested mordants, which have also been used in the light microscopical technique, did not give any useful new information.     

In vivo assessment of emphysema in mice by high resolution X-ray microtomography

High resolution X-ray microtomography (micro-CT) was used for the detection of emphysema in live mice. Emphysema was induced in C57BL/6 J mice by intratracheal instillation of different amounts of porcine pancreatic elastase. This emphysema could be clearly detected by micro-CT seven weeks post-treatment: analysis of the whole data set of virtual cross-sections showed the presence of a dose-dependent level of emphysema.     

香烟燃烧挥发颗粒物的二次离子质谱分析研究(英文)

在室内气氛中,吸烟释放出的颗粒物是倍受关心的污染源之一。本工作运用高性能静态二次离子质谱（TOF SIMS）实验研究了模拟吸烟释放的气溶胶颗粒物中的有机污染物。证实了其中含有氮杂环化合物和多环芳烃。从而初步表明,二次离子质谱在快速表征室内环境污染物方面具有潜在运用价值。     

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.     

Influence of fixative osmolality on the morphometric determination of extracellular space in normal and reperfused ischaemic myocardium

Morphometric determination of extracellular space in control and post-ischaemic reperfused rabbit myocardium was evaluated using two fixatives differing in their composition and total osmolality. Measurement of control extracellular space in an isotonic fixative (294 mOsm/kg water) was 20.8% and in a hypertonic fixative (1816 mOsm/kg water) was 22.2%. These values were not statistically different. Ischaemic durations of 15, 30, 60 and 90 min, followed by an equivalent period of reperfusion, created significant increases in extracellular space. The size of the extracellular space determined by both fixatives was found to be the same. Total fixative osmolality does not appear to influence morphometric evaluation of the extracellular space in control tissue or in tissue damaged by ischaemia and reperfusion.     

基于DEM算法的多颗粒碰撞耗能机理分析与振动抑制研究

针对机械构件主系的封闭空间中填充微小颗粒,进行振动抑制问题,对填充颗粒的尺寸、数量以及材料特性因素对振动抑制效果的影响开展了研究。通过采用离散单元法(discrete element method,DEM),分析了颗粒与颗粒以及颗粒与主系统之间的运动学特性,建立了能够充分表达颗粒在相互碰撞摩擦过程中的受力、变形关系以及耗能计算模型,分析了多个颗粒之间的碰撞与耗能机理,确定了碰撞过程中颗粒的状态、受力及耗能大小的计算求解算法。在Matlab环境下,针对不同颗粒材质、数量及大小对系统振动抑制性能进行了仿真分析,得出了颗粒材质大小以及数量对减振性能的影响规律,并通过搭建的试验台,进行了试验数据采集和分析。试验结果与仿真结果相吻合,验证了算法的有效性,该算法为提高机械构件的减振性能设计提供了重要参考。     

Assessment of particle retention and clearance in the intrapulmonary conducting airways of hamster lungs with the fractionator1

A modified version of the fractionator was used to estimate the total number of polystyrene microspheres retained in the airways of hamster lungs at two different time points after inhalation. A systematic three-stage subsampling procedure with known sampling fractions was adopted. First, each lung was cut into slices, from which primary disectors were sampled systematically with a known sampling fraction. From each primary disector, smaller sub-disectors were subsampled, and the corresponding sampling fraction was estimated by point counting. Finally, a few particles were counted at the microscopic level in the sub-disectors, and the final estimate of total particle number (which is unbiased irrespective of any tissue deformations) was easily computed as a product of the counted number times the reciprocal of the successive sampling fractions. The error variance of each estimate was assessed from the data using a new estimator. An average of 6% of the deposited particles were retained on the epithelial surface of the intrapulmonary conducting airways shortly after the inhalation, from which at least one-third was already phagocytosed by macrophages. After 24 h, an average of 87% of the particles retained shortly after the inhalation had been cleared. The proportion of particles ingested by macrophages had increased to at least 87%, in three out of four animals studied.     

High-resolution scanning electron microscopy of frozen-hydrated and freeze-substituted kidney tissue

Inner surfaces and fracture faces of rabbit kidney tissue were investigated with high-resolution scanning electron microscopy using two different cryopreparation techniques: (i) for the observation of fracture faces, cryofixed tissue was fractured and coated in a cryopreparation chamber dedicated to SEM, vacuum transferred onto a cold stage and observed in the frozen-hydrated state; (ii) for the observation of inner surfaces of the nephron, water was removed after freezing and fracturing by freeze substitution and critical-point drying of the tissue. By both methods, macromolecular structures such as intramembranous particles on fracture faces and particles on inner surfaces were imaged. The latter method was used to investigate in more detail surface structures of cells in the cortical collecting duct. These studies revealed a heterogeneity of intercalated cells not described thus far.     

Velocity measurements in dense down flow of bulk solids using a non-restrictive acoustic method

The velocity of a dense down flow of particles in a conduit can be measured in-line and non-invasively using acoustic waves to detect the passage of characteristic patterns in the local voidage. These patterns are, however, distorted as they flow through the conduit due to the relative motion between the particles, and shear zones at the wall. This work investigates the effect that the particle velocity and acoustic sampling parameters have on the correlation and velocity measurement of these patterns.     

肺磁图的研究进展

肺磁图是一种通过测定铁磁性粉尘的剩余磁场来衡量肺部粉尘含量的方法,具有无损伤、灵敏度高、可重复性好等优点。根据世界各国最近三十年在该领域的研究进展,本文从肺磁图原理、技术方法、肺磁信号影响因素这三个方面做出系统归纳,并结合相关实践进展,指出肺磁图技术在肺部粉尘估算、肺功能状态评价及细胞生理特性研究领域的应用。     

HREM and STEM of defects in multiply-twinned particles

The structure of defects in multiply-twinned particles has been studied in detail using high-resolution lattice imaging, dark field and microdiffraction techniques. Icosahedral particles with sizes greater than about 15 nm were found to contain defects, in the form of stacking fault loops parallel with the external surface, which were extremely difficult to detect by conventional amplitude contrast techniques. Microdiffraction mappings correlated with these results, showing large rotations of the face-centred cubic segments. Results for decahedral particles indicated the presence of stacking faults running adjacent to, and parallel with, the twin boundaries. Microdiffraction maps confirmed that the particle structure was face-centred cubic, and also verified that the apparent epitaxy of these particles was highly variable. Models for the defects are proposed and discussed, and the relative merits of HREM and STEM for elucidating structural details in small particles is briefly considered. Finally, the potential for direct imaging at surfaces, as demonstrated by some recent images, is discussed.     

Three-dimensional investigation and scoring of extracellular matrix remodeling during lung fibrosis using multiphoton microscopy

The organization of collagen during fibrotic processes is poorly characterized because of the lack of appropriate methodologies. Here we show that multimodal multiphoton microscopy provides novel insights into lung fibrosis. We characterize normal and fibrotic pulmonary tissue in the bleomycin model, and show that second-harmonic generation by fibrillar collagen reveals the micrometer-scale three-dimensional spatial distribution of the fibrosis. We find that combined two-photon excited fluorescence and second-harmonic imaging of unstained lung tissue allows separating the inflammatory and fibrotic steps in this pathology, underlining characteristic features of fibroblastic foci in human Idiopathic Pulmonary Fibrosis samples. Finally, we propose phenomenological scores of lung fibrosis and we show that they unambiguously sort out control and treated mice, with a better sensitivity and reproducibility in the subpleural region. These results should be readily generalized to other organs, as an accurate method to assess extracellular matrix remodeling during fibrosis.     

Moiré fringe analysis of small precipitates in melt-spun titanium-silicon alloys

Oxygen-contaminated, melt-spun, binary Ti-Si alloys have been examined by using transmission electron microscopy. The microstructure of alloys in the range of 4 to 10% Si (by weight) are cellular and consist primarily of α-Ti and the silicide Ti₅Si_3. Contained only within the Ti₅Si₃regions are small, approximately spherical particles which are ? 10 nm in diameter. Due to their small size, the crystal structure of these particles could not be determined by using conventional diffraction techniques such as Selected Area or Convergent Beam Diffraction. By conducting a number of tilting experiments and observing the moire fringe patterns produced when various matrixTi₅Si₃ planes were used to image the sample, the crystal structure of the particles and the orientation relationship which exists between them and the matrix were deduced. The unknown particles, termed the Z phase, were found to be hexagonal with slightly different lattice parameters from the matrix Ti₅Si_3. Their relationship with the matrix was such that they appeared to be totally coherent. This may indicate that Z is an oxide based on the intermetallic Ti₅Si₃.     

Application of confocal scanning laser microscopy in experimental pathology

Confocal scanning laser microscopy (CSLM) represents an exciting new tool for scientific disciplines which focus on mechanistic studies such as experimental pathology. Enhanced resolution in the specimen plane and rejection of out-of-focus fluorescence flare allow analysis of specific nucleic acid sequences, enzymes, structural macromolecules, and cellular homeostasis utilizing fluorescent probes. Four different experimental applications are discussed which utilize CSLM to evaluate pathological processes at the subcellular, cellular, and tissue levels. Programmed cell death, or apoptosis, is a natural process of significance both during development and as a response to toxic stimuli. CSLM-imaging of nuclei of human B lymphoblastoid cells following exposure to a monofunctional alkylating agent suggests that the degradation of chromatin characteristic of apoptosis may occur in asymmetric patterns. Surfactant apoprotein-A is the major non-serum protein component of pulmonary surfactant and is essential for the extracellular function of surfactant. CSLM of alveolar type II cells suggests that apoprotein-A is present in both the cytoplasm, predominantly in lamellar bodies, and in the nucleus. The tumor promoter, phorbol myristate acetate, rapidly stimulated the formation of vacuoles in human neutrophils. CSLM using Lucifer Yellow as a probe suggests that cylindrical vacuoles are formed by fluid-phase pinocytosis. The blood-nerve barrier (BNB) in peripheral nerves may be an important target during toxin-induced neuropathies. Ricin-induced permeability of the BNB in the rat was rapidly visualized by CSLM as leakage of fluorescein isothiocynate (FITC)-dextran into the endoneurial compartment.     

Statistical modelling of the geometry of planar sections of prostatic capillaries on the basis of stationary Strauss hard-core processes

In a recent study, the capillarization of normal prostatic tissue and prostatic carcinoma tissue was characterized by means of explorative methods of spatial statistics. In the present paper, an attempt was made to go beyond the explorative approach and to characterize the observed point patterns of the capillary profiles on sections by means of a parametric model. For this purpose, the flexible class of Gibbs processes was considered. Specifically, stationary Strauss hard-core processes were fitted to the observed point patterns. The goodness of fit achieved by the model was checked by simulations with the Markov chain Monte Carlo method using the Metropolis–Hastings algorithm. Model fitting and simulations were performed with the help of the spatstat package under R. The observed point patterns were in some cases compatible with realizations of stationary Strauss hard-core processes for all ranges of spatial interaction. However, deviations from the model were found for one or more domains of ranges in other cases. In the tumour tissue, a highly significant decrease of the interaction parameter of the Strauss hard-core process could be found as compared to the normal prostatic tissue. This finding is discussed in terms of a loss of the normal lobular architecture of the glands in the tumour tissue.     

Cryopreparation of mammalian tissue for X-ray microanalysis in STEM

A cryopreparation technique for studies of ultrastructure and distribution of diffusible elements in biological tissue is described. Electron microscopical contrast and characteristic X-ray spectra are found to be poor in completely frozen-hydrated ultrathin cryosections of fresh chemically untreated tissue. Both STEM contrast and detection of characteristic X-rays are enhanced by careful freeze-drying in the microscope. Although the ultrastructure is affected by ice crystals, intracellular compartments can be identified by STEM without staining and studied by X-ray microanalysis.     

Rapid and reliable method for diagnostic electron microscopy of faeces

Four methods are described for examining viruses in faeces by electron microscopy using negative staining. Faeces samples from a total of 180 patients with diarrhoea illnesses were processed for electron microscopy by the direct staining technique, pseudoreplica technique, microsolute concentration technique and by ultracentrifugation. Virus particles (including rotaviruses and adenoviruses) were found in fifty-five (31%) samples when the results of all methods were combined. Bacteriophages were found in thirty of the samples which were read virus negative. Herpesviruses were also found in the faecal samples of two patients with diarrhoea illness. Parvovirus-like particles were identified in one sample which was rotavirus and adeno-virus positive. Calicivirus-like particles were found in a sample which was adenovirus positive. With the direct staining technique thirty-six (20%) samples contain virus particles; with micro-solute concentration technique forty-eight (27%) samples contain virus particles and with ultracentrifugation thirty-eight (21%) contain virus particles. It is believed the microsolute concentration technique is rapid and more reliable than the pseudoreplica technique and ultracentrifugation method, and is a more preferable method for the diagnosis of faecal sample by electron microscopy.