Real time analysis of atmospheric single particles in urban environments using aerosol time of flight mass spectrometry

In order to determine the size-resolved chemical composition of single particles in real-time an ATOFMS was deployed at urban background sites in Paris and Barcelona during the MEGAPOLI and SAPUSS monitoring campaigns respectively. The particle types detected during MEGAPOLI included several carbonaceous species, metal-containing types and sea-salt. Elemental carbon particle types were highly abundant, with 86% due to fossil fuel combustion and 14% attributed to biomass burning. Furthermore, 79% of the EC was apportioned to local emissions and 21% to continental transport. The carbonaceous particle types were compared with quantitative measurements from other instruments, and while direct correlations using particle counts were poor, scaling of the ATOFMS counts greatly improved the relationship. During SAPUSS carbonaceous species, sea-salt, dust, vegetative debris and various metal-containing particle types were identified. Throughout the campaign the site was influenced by air masses altering the composition of particles detected. During North African air masses the city was heavily influenced by Saharan dust. A regional stagnation was also observed leading to a large increase in carbonaceous particle counts. While the ATOFMS provides a list of particle types present during the measurement campaigns, the data presented is not directly quantitative. The quantitative response of the ATOFMS to metals was examined by comparing the ion signals within particle mass spectra and to hourly mass concentrations of; Na, K, Ca, Ti, V, Cr, Mn, Fe, Zn and Pb. The ATOFMS was found to have varying correlations with these metals depending on sampling issues such as matrix effects. The strongest correlations were observed for Al, Fe, Zn, Mn and Pb. Overall the results of this work highlight the excellent ability of the ATOFMS in providing composition and mixing state information on atmospheric particles at high time resolution. However they also show its limitations in delivering quantitative information directly.

Beyond simple imaging with energetic beams – nanopatterning, correlative microscopy and statistical analysis of inclusions

Electron microscopy (EM) has advanced in an exponential way since the first transmission electron microscope (TEM) was built in the 1930’s. The urge to ‘see’ things is an essential part of human nature (talk of ‘seeing is believing’) and apart from scanning tunnel microscopes which give information about the surface, EM is the only imaging technology capable of really visualising atomic structures in depth down to single atoms. With the development of nanotechnology the demand to image and analyse small things has become even greater and electron microscopes have found their way from highly delicate and sophisticated research grade instruments to key-turn and even bench-top instruments for everyday use in every materials research lab on the planet. The semiconductor industry is as dependent on the use of EM as life sciences and pharmaceutical industry. With this generalisation of use for imaging, the need to deploy advanced uses of EM has become more and more apparent. The combination of several coinciding beams (electron, ion and even light) to create DualBeam or TripleBeam instruments for instance enhances the usefulness from pure imaging to manipulating on the nanoscale. And when it comes to the analytic power of EM with the many ways the highly energetic electrons and ions interact with the matter in the specimen there is a plethora of niches which evolved during the last two decades, specialising in every kind of analysis that can be thought of and combined with EM. In the course of this study the emphasis was placed on the application of these advanced analytical EM techniques in the context of multiscale and multimodal microscopy – multiscale meaning across length scales from micrometres or larger to nanometres, multimodal meaning numerous techniques applied to the same sample volume in a correlative manner. In order to demonstrate the breadth and potential of the multiscale and multimodal concept an integration of it was attempted in two areas: I) Biocompatible materials using polycrystalline stainless steel and II) Semiconductors using thin multiferroic films. I) The motivation to use stainless steel (316L medical grade) comes from the potential modulation of endothelial cell growth which can have a big impact on the improvement of cardio-vascular stents – which are mainly made of 316L – through nano-texturing of the stent surface by focused ion beam (FIB) lithography. Patterning with FIB has never been reported before in connection with stents and cell growth and in order to gain a better understanding of the beam-substrate interaction during patterning a correlative microscopy approach was used to illuminate the patterning process from many possible angles. Electron backscattering diffraction (EBSD) was used to analyse the crystallographic structure, FIB was used for the patterning and simultaneously visualising the crystal structure as part of the monitoring process, scanning electron microscopy (SEM) and atomic force microscopy (AFM) were employed to analyse the topography and the final step being 3D visualisation through serial FIB/SEM sectioning. II) The motivation for the use of thin multiferroic films stems from the ever-growing demand for increased data storage at lesser and lesser energy consumption. The Aurivillius phase material used in this study has a high potential in this area. Yet it is necessary to show clearly that the film is really multiferroic and no second phase inclusions are present even at very low concentrations – ~0.1vol% could already be problematic. Thus, in this study a technique was developed to analyse ultra-low density inclusions in thin multiferroic films down to concentrations of 0.01%. The goal achieved was a complete structural and compositional analysis of the films which required identification of second phase inclusions (through elemental analysis EDX(Energy Dispersive X-ray)), localise them (employing 72 hour EDX mapping in the SEM), isolate them for the TEM (using FIB) and give an upper confidence limit of 99.5% to the influence of the inclusions on the magnetic behaviour of the main phase (statistical analysis).

Probing polarization and dielectric function of molecules with higher order harmonics in scattering-near-field scanning optical microscopy

The idealized system of an atomically flat metallic surface [highly oriented pyrolytic graphite (HOPG)] and an organic monolayer (porphyrin) was used to determine whether the dielectric function and associated properties of thin films can be accessed with scanning-near-field scanning optical microscopy (s-NSOM). Here, we demonstrate the use of harmonics up to fourth order and the polarization dependence of incident light to probe dielectric properties on idealized samples of monolayers of organic molecules on atomically smooth substrates. An analytical treatment of light/sample interaction using the s-NSOM tip was developed in order to quantify the dielectric properties. The theoretical analysis and numerical modeling, as well as experimental data, demonstrate that higher order harmonic scattering can be used to extract the dielectric properties of materials with tens of nanometer spatial resolution. To date, the third harmonic provides the best lateral resolution (∼50 nm) and dielectric constant contrast for a porphyrin film on HOPG. © 2009 American Institute of Physics.

Separating DNA with different topologies by atomic force microscopy in comparison with gel electrophoresis.

Atomic force microscopy, which is normally used for DNA imaging to gain qualitative results, can also be used for quantitative DNA research, at a single-molecular level. Here, we evaluate the performance of AFM imaging specifically for quantifying supercoiled and relaxed plasmid DNA fractions within a mixture, and compare the results with the bulk material analysis method, gel electrophoresis. The advantages and shortcomings of both methods are discussed in detail. Gel electrophoresis is a quick and well-established quantification method. However, it requires a large amount of DNA, and needs to be carefully calibrated for even slightly different experimental conditions for accurate quantification. AFM imaging is accurate, in that single DNA molecules in different conformations can be seen and counted. When used carefully with necessary correction, both methods provide consistent results. Thus, AFM imaging can be used for DNA quantification, as an alternative to gel electrophoresis.

Simultaneous two-wavelength transmission quantitative phase microscopy with a color camera.

We present a quantitative phase microscopy method that uses a Bayer mosaic color camera to simultaneously acquire off-axis interferograms in transmission mode at two distinct wavelengths. Wrapped phase information is processed using a two-wavelength algorithm to extend the range of the optical path delay measurements that can be detected using a single temporal acquisition. We experimentally demonstrate this technique by acquiring the phase profiles of optically clear microstructures without 2pi ambiguities. In addition, the phase noise contribution arising from spectral channel crosstalk on the color camera is quantified.

A versatile diffractive maskless lithography for single-shot and serial microfabrication.

We demonstrate a diffractive maskless lithographic system that is capable of rapidly performing both serial and single-shot micropatterning. Utilizing the diffractive properties of phase holograms displayed on a spatial light modulator, arbitrary intensity distributions were produced to form two and three dimensional micropatterns/structures in a variety of substrates. A straightforward graphical user interface was implemented to allow users to load templates and change patterning modes within the span of a few minutes. A minimum resolution of approximately 700 nm is demonstrated for both patterning modes, which compares favorably to the 232 nm resolution limit predicted by the Rayleigh criterion. The presented method is rapid and adaptable, allowing for the parallel fabrication of microstructures in photoresist as well as the fabrication of protein microstructures that retain functional activity.

Pulsating tandem microbubble for localized and directional single-cell membrane poration.

The interaction of laser-generated tandem microbubble (maximum diameter of about 50  μm) with single (rat mammary carcinoma) cells is investigated in a 25-μm liquid layer. Antiphase and coupled oscillation of the tandem microbubble leads to the formation of alternating, directional microjets (with max microstreaming velocity of 10  m/s) and vortices (max vorticity of 350 000  s{-1}) in opposite directions. Localized and directional membrane poration (200 nm to 2  μm in pore size) can be produced by the tandem microbubble in an orientation and proximity-dependent manner, which is absent from a single oscillating microbubble of comparable size and at the same stand-off distance.

Explicit DNase sequence bias modeling enables high-resolution transcription factor footprint detection.

DNaseI footprinting is an established assay for identifying transcription factor (TF)-DNA interactions with single base pair resolution. High-throughput DNase-seq assays have recently been used to detect in vivo DNase footprints across the genome. Multiple computational approaches have been developed to identify DNase-seq footprints as predictors of TF binding. However, recent studies have pointed to a substantial cleavage bias of DNase and its negative impact on predictive performance of footprinting. To assess the potential for using DNase-seq to identify individual binding sites, we performed DNase-seq on deproteinized genomic DNA and determined sequence cleavage bias. This allowed us to build bias corrected and TF-specific footprint models. The predictive performance of these models demonstrated that predicted footprints corresponded to high-confidence TF-DNA interactions. DNase-seq footprints were absent under a fraction of ChIP-seq peaks, which we show to be indicative of weaker binding, indirect TF-DNA interactions or possible ChIP artifacts. The modeling approach was also able to detect variation in the consensus motifs that TFs bind to. Finally, cell type specific footprints were detected within DNase hypersensitive sites that are present in multiple cell types, further supporting that footprints can identify changes in TF binding that are not detectable using other strategies.

High-Resolution CT for Small-Animal Imaging Research

Preclinical imaging has a critical role in phenotyping, in drug discovery, and in providing a basic understanding of mechanisms of disease. Translating imaging methods from humans to small animals is not an easy task. The purpose of this work is to review high-resolution computed tomography (CT) also known as micro-CT for small-animal imaging. We present the principles, the technologies, the image quality parameters, and the types of applications. We show that micro-CT can be used to provide not only morphological but also functional information such as cardiac function or vascular permeability. Another way in which micro-CT can be used in the study of both function and anatomy is by combining it with other imaging modalities, such as positron emission tomography or single-photon emission tomography. Compared to other modalities, micro-CT imaging is usually regarded as being able to provide higher throughput at lower cost and higher resolution. The limitations are usually associated with the relatively poor contrast mechanisms and the radiation damage, although the use of novel nanoparticle-based contrast agents and careful design of studies can address these limitations.

New data inversion formulae in confocal scanning microscopy

Whereas the resolving power of an ordinary optical microscope is determined by the classical Rayleigh distance, significant super-resolution, i.e. resolution improvement beyond that Rayleigh limit, has been achieved by confocal scanning light microscopy. Furthermore is has been shown that the resolution of a confocal scanning microscope can still be significantly enhanced by measuring, for each scanning position, the full diffraction image by means of an array of detectors and by inverting these data to recover the value of the object at the focus. We discuss the associated inverse problem and show how to generalize the data inversion procedure by allowing, for reconstructing the object at a given point, to make use also of the diffraction images recorded at other scanning positions. This leads us to a whole family of generalized inversion formulae, which contains as special cases some previously known formulae. We also show how these exact inversion formulae can be implemented in practice.

Infrared light on molecule-molecule and molecule-surface collisions

By analyzing measured infrared absorption of pure CH4 gas under both "free" (large sample cell) and "confined" (inside the pores of a silica xerogel sample) conditions we give a demonstration that molecule-molecule and molecule-surface collisions lead to very different propensity rules for rotational-state changes. Whereas the efficiency of collisions to change the rotational state (observed through the broadening of the absorption lines) decreases with increasing rotational quantum number J for CH4-CH4 interactions, CH4-surface collisions lead to J-independent linewidths. In the former case, some (weak) collisions are inefficient whereas, in the latter case, a single collision is sufficient to remove the molecule from its initial rotational level. Furthermore, although some gas-phase collisions leave J unchanged and only modify the angular momentum orientation and/or symmetry of the level (as observed through the spectral effects of line mixing), this is not the case for the molecule-surface collisions since they always change J (in the studied J=0-14 range).

Genetically monomorphic brown trout (Salmo trutta L.) populations as revealed by mitochondrial DNA, multi-locus and single-locus minisatellite (VNTR) analyses.

Normally, populations of brown trout are genetically highly variable. Two adjacent populations from NW Scotland, which had previously been found to be monomorphic for 46 protein-coding loci, were studied by higher resolution techniques. Analyses of mitochondrial DNA, multilocus DNA fingerprints and eight specific minisatellite loci revealed no genetic variation among individuals or genetic differences between the two populations. Continual low effective population sizes or severe repeated bottlenecks, as a result of low or variable recruitment, probably explain the atypical absence of genetic variation in these trout populations. Growth data do not provide any evidence of a reduction in fitness in trout from these populations.

Differences in expression of junctional adhesion molecule-A and beta catenin in multiple sclerosis brain tissue: increasing evidence for the role of tight junction pathology

Previously we have employed antibodies to the tight junction (TJ)-associated proteins ZO-1 and occludin to describe endothelial tight junction abnormalities, in lesional and normal appearing white matter, in primary and secondary progressive multiple sclerosis (MS). This work is extended here by use of antibodies to the independent TJ-specific proteins and junctional adhesion molecule A & B (JAM-A, JAM-B). We have also assessed the expression in MS of ß-catenin, a protein specific to the TJ-associated adherens junction. Immunocytochemistry and semiquantitative confocal microscopy for JAM-A and ß-catenin was performed on snap-frozen sections from MS cases (n = 11) and controls (n = 6). Data on 1,443 blood vessels was acquired from active lesions (n = 13), inactive lesions (n = 13), NAWM (n = 20) and control white matter (n = 13). In MS abnormal JAM-A expression was found in active (46%) and inactive lesions (21%), comparable to previous data using ZO-1. However, a lower level of TJ abnormality was found in MS NAWM using JAM-A (3%) compared to ZO-1 (13%). JAM-B was strongly expressed on a small number of large blood vessels in control and MS tissues but at too low a level for quantitative analysis. By comparison with the high levels of abnormality observed with the TJ proteins, the adherens junction protein ß-catenin was normally expressed in all MS and control tissue categories. These results confirm, by use of the independent marker JAM-A, that TJ abnormalities are most frequent in active white matter lesions. Altered expression of JAM-A, in addition to affecting junctional tightness may also both reflect and affect leukocyte trafficking, with implications for immune status within the diseased CNS. Conversely, the adherens junction component of the TJ, as indicated by ß-catenin expression is normally expressed in all MS and control tissue categories.

Triclabendazole-resistant Fasciola hepatica: β-tubulin and response to in vitro treatment with triclabendazole

Resistance in Fasciola hepatica to triclabendazole (Fasinex) has emerged in several countries. Benzimidazole resistance in parasitic nematodes has been linked to a single amino acid substitution (phenylalanine to tyrosine) at position 200 on the [beta]-tubulin molecule. Sequencing of [beta]-tubulin cDNAs from triclabendazole-susceptible and triclabendazole-resistant flukes revealed no amino acid differences between their respective primary amino acid sequences. In order to investigate the mechanism of triclabendazole resistance, triclabendazole-susceptible and triclabendazole-resistant flukes were incubated in vitro with triclabendazole sulphoxide (50 [mu]g/ml). Scanning and transmission electron microscopy revealed extensive damage to the tegument of triclabendazole-susceptible F. hepatica, whereas triclabendazole-resistant flukes showed only localized and relatively minor disruption of the tegument covering the spines. Immunocytochemical studies, using an anti-tubulin antibody, showed that tubulin organization was disrupted in the tegument of triclabendazole-susceptible flukes. No such disruption was evident in triclabendazole-resistant F. hepatica. The significance of these findings is discussed with regard to the mechanism of triclabendazole resistance in F. hepatica.