Electron energy-loss spectroscopy as a tool for elemental analysis in biological speciments.

A transmission electron microscope (TEM) accessory, the energy filter, enables the establishment of a method for elemental microanalysis, the electron energy-loss spectroscopy (EELS). In conventional TEM, unscattered, elastic, and inelastic scattered electrons contribute to image information. Energy-filtering TEM (EFTEM) allows elemental analysis at the ultrastructural level by using selected inelastic scattered electrons. EELS is an excellent method for elemental microanalysis and nanoanalysis with good sensitivity and accuracy. However, it is a complex method whose potential is seldom completely exploited, especially for biological specimens. In addition to spectral analysis, parallel-EELS, we present two different imaging techniques in this chapter, namely electron spectroscopic imaging (ESI) and image-EELS. We aim to introduce these techniques in this chapter with the elemental microanalysis of titanium. Ultrafine, 22-nm titanium dioxide particles are used in an inhalation study in rats to investigate the distribution of nanoparticles in lung tissue.

Microgram level radiocarbon (14C) determination on carbonaceous particles in ice

In climate research the interest on carbonaceous particles has increased over the last years because of their influence on the radiation balance of the earth. Nevertheless, there is a paucity of available data regarding their concentrations and sources in the past. Such data would be important for a better understanding of their effects and for estimating their influence on future climate. Here, a technique is described to extract carbonaceous particles from ice core samples with subsequent separation of the two main constituents into organic carbon (OC) and elemental carbon (EC) for analysis of their concentrations in the past. This is combined with further analysis of OC and EC 14C/12C ratios by accelerator mass spectrometry (AMS), what can be used for source apportionment studies of past emissions. We further present how 14C analysis of the OC fraction could be used in the future to date any ice core extracted from a high-elevation glacier. Described sample preparation steps to final analysis include the combustion of micrograms of water–insoluble carbonaceous particles, primary collected by filtration of melted ice samples, the graphitisation of the obtained CO2 to solid AMS target material and final AMS measurements. Possible fractionation processes were investigated for quality assurance. Procedural blanks were reproducible and resulted in carbon masses of 1.3 ± 0.6 μg OC and 0.3 ± 0.1 μg EC per filter. The determined fraction of modern carbon (fM) for the OC blank was 0.61 ± 0.13. The analysis of processed IAEA-C6 and IAEA-C7 reference material resulted in fM = 1.521 ± 0.011 and δ13C = −10.85 ± 0.19‰, and fM = 0.505 ± 0.011 and δ13C = −14.21 ± 0.19‰, respectively, in agreement with consensus values. Initial carbon contents were thereby recovered with an average yield of 93%.

A novel quantitative method for analyzing the distributions of nanoparticles between different tissue and intracellular compartments

The penetration, translocation, and distribution of ultrafine and nanoparticles in tissues and cells are challenging issues in aerosol research. This article describes a set of novel quantitative microscopic methods for evaluating particle distributions within sectional images of tissues and cells by addressing the following questions: (1) is the observed distribution of particles between spatial compartments random? (2) Which compartments are preferentially targeted by particles? and (3) Does the observed particle distribution shift between different experimental groups? Each of these questions can be addressed by testing an appropriate null hypothesis. The methods all require observed particle distributions to be estimated by counting the number of particles associated with each defined compartment. For studying preferential labeling of compartments, the size of each of the compartments must also be estimated by counting the number of points of a randomly superimposed test grid that hit the different compartments. The latter provides information about the particle distribution that would be expected if the particles were randomly distributed, that is, the expected number of particles. From these data, we can calculate a relative deposition index (RDI) by dividing the observed number of particles by the expected number of particles. The RDI indicates whether the observed number of particles corresponds to that predicted solely by compartment size (for which RDI = 1). Within one group, the observed and expected particle distributions are compared by chi-squared analysis. The total chi-squared value indicates whether an observed distribution is random. If not, the partial chi-squared values help to identify those compartments that are preferential targets of the particles (RDI > 1). Particle distributions between different groups can be compared in a similar way by contingency table analysis. We first describe the preconditions and the way to implement these methods, then provide three worked examples, and finally discuss the advantages, pitfalls, and limitations of this method.

Translocation of particles and inflammatory responses after exposure to fine particles and nanoparticles in an epithelial airway model

ABSTRACT: BACKGROUND: Experimental studies provide evidence that inhaled nanoparticles may translocate over the airspace epithelium and cause increased cellular inflammation. Little is known, however, about the dependence of particle size or material on translocation characteristics, inflammatory response and intracellular localization. RESULTS: Using a triple cell co-culture model of the human airway wall composed of epithelial cells, macrophages and dendritic cells we quantified the entering of fine (1 mum) and nano-sized (0.078 mum) polystyrene particles by laser scanning microscopy. The number distribution of particles within the cell types was significantly different between fine and nano-sized particles suggesting different translocation characteristics. Analysis of the intracellular localization of gold (0.025 mum) and titanium dioxide (0.02-0.03 mum) nanoparticles by energy filtering transmission electron microscopy showed differences in intracellular localization depending on particle composition. Titanium dioxide nanoparticles were detected as single particles without membranes as well as in membrane-bound agglomerations. Gold nanoparticles were found inside the cells as free particles only. The potential of the different particle types (different sizes and different materials) to induce a cellular response was determined by measurements of the tumour necrosis factor-alpha in the supernatants. We measured a 2-3 fold increase of tumour necrosis factor-alpha in the supernatants after applying 1 mum polystyrene particles, gold nanoparticles, but not with polystyrene and titanium dioxide nanoparticles. CONCLUSION: Quantitative laser scanning microscopy provided evidence that the translocation and entering characteristics of particles are size-dependent. Energy filtering transmission electron microscopy showed that the intracellular localization of nanoparticles depends on the particle material. Both particle size and material affect the cellular responses to particle exposure as measured by the generation of tumour necrosis factor-alpha.

Gunshot-related displacement of skin particles and bacteria from the exit region back into the bullet path

In previous studies, it was shown that there is a gunshot-related transport of skin particles and microorganisms from the entrance region into the depth of the bullet path. The present study deals with the question of whether gunshots may also cause a retrograde transport of skin particles and microorganisms from the bullet exit region back into the bullet path. For this purpose, we used a composite model consisting of rectangular gelatin blocks and pig skin. The skin pieces were firmly attached to the gelatin blocks on the side where the bullet was to exit. Prior to the test shots, the outer surface of the pig skin was contaminated with a thin layer of a defined bacterial suspension. After drying the skin, test shots were fired from a distance of 10 m using cartridges calibre .38 spec. with different bullet types. Subsequent analyses showed that in all shots with full penetration of the composite model, the bullet path contained displaced skin particles and microorganisms from the skin surface at the exit site. These could be regularly detected in the distal 6-8 cm of the track, occasionally up to a distance of 18 cm from the exit hole. The distribution of skin particles and microorganisms is presented and the possible mechanism of this retrograde transport is discussed.

A novel exposure system for the efficient and controlled deposition of aerosol particles onto cell cultures

Epidemiologic studies have shown correlations between morbidity and particles < or = 2.5 microm generated from pollution processes and manufactured nanoparticles. Thereby nanoparticles seem to play a specific role. The interaction of particles with the lung, the main pathway of undesired particle uptake, is poorly understood. In most studies investigating these interactions in vitro, particle deposition differs greatly from the in vivo situation, causing controversial results. We present a nanoparticle deposition chamber to expose lung cells mimicking closely the particle deposition conditions in the lung. In this new deposition chamber, particles are deposited very efficiently, reproducibly, and uniformly onto the cell culture, a key aspect if cell responses are quantified in respect to the deposited particle number. In situ analyses of the lung cells, e.g., the ciliary beat frequency, indicative of the defense capability of the cells, are complemented by off-line biochemical, physiological, and morphological cell analyses.

Combined ultrasmall superparamagnetic particles of iron oxide-enhanced and diffusion-weighted magnetic resonance imaging reliably detect pelvic lymph node metastases in normal-sized nodes of bladder and prostate cancer patients

BACKGROUND: Lymph node staging of bladder or prostate cancer using conventional imaging is limited. Newer approaches such as ultrasmall superparamagnetic particles of iron oxide (USPIO) and diffusion-weighted magnetic resonance imaging (DW-MRI) have inconsistent diagnostic accuracy and are difficult to interpret. OBJECTIVE: To assess whether combined USPIO and DW-MRI (USPIO-DW-MRI) improves staging of normal-sized lymph nodes in bladder and/or prostate cancer patients. DESIGN, SETTING, AND PARTICIPANTS: Twenty-one consecutive patients with bladder and/or prostate cancer were enrolled between May and October 2008. One patient was excluded secondary to bone metastases detected on DW-MRI with subsequent abstention from surgery. INTERVENTION: Patients preoperatively underwent 3-T MRI before and after administration of lymphotropic USPIO using conventional MRI sequences combined with DW-MRI. Surgery consisted of extended pelvic lymphadenectomy and resection of primary tumors. MEASUREMENTS: Diagnostic accuracies of the new combined USPIO-DW-MRI approach compared with the "classic" reading method evaluating USPIO images without and with DW-MRI versus histopathology were evaluated. Duration of the two reading methods was noted for each patient. RESULTS AND LIMITATIONS: Diagnostic accuracy (90% per patient or per pelvic side) was comparable for the classic and the USPIO-DW-MRI reading method, while time of analysis with 80 min (range 45-180 min) for the classic and 13 min (range 5-90 min) for the USPIO-DW-MRI method was significantly shorter (p<0.0001). Interobserver agreement (three blinded readers) was high with a kappa value of 0.75 and 0.84, respectively. Histopathological analysis showed metastases in 26 of 802 analyzed lymph nodes (3.2%). Of these, 24 nodes (92%) were correctly diagnosed as positive on USPIO-DW-MRI. In two patients, one micrometastasis each (1.0x0.2 mm; 0.7x0.4 mm) was missed in all imaging studies. CONCLUSIONS: USPIO-DW-MRI is a fast and accurate method for detecting pelvic lymph node metastases, even in normal-sized nodes of bladder or prostate cancer patients.

Diesel exhaust particles modulate the tight junction protein occludin in lung cells in vitro

ABSTRACT: BACKGROUND: Using an in vitro triple cell co-culture model consisting of human epithelial cells (16HBE14o-), monocyte-derived macrophages and dendritic cells, it was recently demonstrated that macrophages and dendritic cells create a transepithelial network between the epithelial cells to capture antigens without disrupting the epithelial tightness. The expression of the different tight junction proteins in macrophages and dendritic cells, and the formation of tight junction-like structures with epithelial cells has been demonstrated. Immunofluorescent methods combined with laser scanning microscopy and quantitative real-time polymerase chain reaction were used to investigate if exposure to diesel exhaust particles (DEP) (0.5, 5, 50, 125 mug/ml), for 24 h, can modulate the expression of the tight junction mRNA/protein of occludin, in all three cell types. RESULTS: Only the highest dose of DEP (125 mug/ml) seemed to reduce the occludin mRNA in the cells of the defence system however not in epithelial cells, although the occludin arrangement in the latter cell type was disrupted. The transepithelial electrical resistance was reduced in epithelial cell mono-cultures but not in the triple cell co-cultures, following exposure to high DEP concentration. Cytotoxicity was not found, in either epithelial mono-cultures nor in triple cell co-cultures, after exposure to the different DEP concentrations. CONCLUSION: We concluded that high concentrations of DEP (125 mug/ml) can modulate the tight junction occludin mRNA in the cells of the defence system and that those cells play an important role maintaining the epithelial integrity following exposure to particulate antigens in lung cells.

Toxic effects of brake wear particles on epithelial lung cells in vitro

ABSTRACT: BACKGROUND: Fine particulate matter originating from traffic correlates with increased morbidity and mortality. An important source of traffic particles is brake wear of cars which contributes up to 20% of the total traffic emissions. The aim of this study was to evaluate potential toxicological effects of human epithelial lung cells exposed to freshly generated brake wear particles. RESULTS: An exposure box was mounted around a car's braking system. Lung cells cultured at the air-liquid interface were then exposed to particles emitted from two typical braking behaviours ("full stop" and "normal deceleration"). The particle size distribution as well as the brake emission components like metals and carbons was measured on-line, and the particles deposited on grids for transmission electron microscopy were counted. The tight junction arrangement was observed by laser scanning microscopy. Cellular responses were assessed by measurement of lactate dehydrogenase (cytotoxicity), by investigating the production of reactive oxidative species and the release of the pro-inflammatory mediator interleukin-8. The tight junction protein occludin density decreased significantly (p < 0.05) with increasing concentrations of metals on the particles (iron, copper and manganese, which were all strongly correlated with each other). Occludin was also negatively correlated with the intensity of reactive oxidative species. The concentrations of interleukin-8 were significantly correlated with increasing organic carbon concentrations. No correlation was observed between occludin and interleukin-8, nor between reactive oxidative species and interleukin-8. CONCLUSION: These findings suggest that the metals on brake wear particles damage tight junctions with a mechanism involving oxidative stress. Brake wear particles also increase pro-inflammatory responses. However, this might be due to another mechanism than via oxidative stress.

Wear particles and surface topographies are modulators of osteoclastogenesis in vitro

Prosthetic and osteosynthetic implants from metal alloys will be indispensable in orthopedic surgery, as long as tissue engineering and biodegradable bone substitutes do not lead to products that will be applied in clinical routine for the repair of bone, cartilage, and joint defects. Therefore, the elucidation of the interactions between the periprosthetic tissues and the implant remains of clinical relevance and several factors are known to affect the longevity of implants. Within this study, the effects of metal particles and surface topography on the recruitment of osteoclasts was investigated in vitro in a coculture of osteoblasts and bone marrow cells. The cells were grown in the presence of particles of different sizes and chemical composition or on metal discs with polished or sandblasted surfaces, respectively. At the end of the culture, newly formed osteoclasts were counted. Osteoclastogenesis was reduced when particles were added directly to the coculture. The effect depended on the size of the particles, small particles exerting stronger effects than larger ones. The chemical composition of the particles, however, did not affect the development of osteoclasts. In cocultures grown on sandblasted surfaces, osteoclasts developed at higher rates than they did in cultures on polished surfaces. The data demonstrate that wear particles and implant surfaces affect osteoclastogenesis and thus may be involved in the induction of local bone resorption and the formation of osteolytic lesions, leading eventually to the loosening of orthopedic implants.