A Bayesian approach to tracking in single molecule fluorescence microscopy

Using fluorescence microscopy with single molecule sensitivity it is now possible to follow the movement of individual fluorophore tagged molecules such as proteins and lipids in the cell membrane with nanometer precision. These experiments are important as they allow many key biological processes on the cell membrane and in the cell, such as transcription, translation and DNA replication, to be studied at new levels of detail. Computerized microscopes generate sequences of images (in the order of tens to hundreds) of the molecules diffusing and one of the challenges is to track these molecules to obtain reliable statistics such as speed distributions, diffusion patterns, intracellular positioning, etc. The data set is challenging because the molecules are tagged with a single or small number of fluorophores, which makes it difficult to distinguish them from the background, the fluorophore bleaches irreversibly over time, the number of tagged molecules are unknown and there is occasional loss of signal from the tagged molecules. All these factors make accurate tracking over long trajectories difficult. Also the experiments are technically difficulty to conduct and thus there is a pressing need to develop better algorithms to extract the maximum information from the data. For this purpose we propose a Bayesian approach and apply our technique to synthetic and a real experimental data set.

Intracellular demography and the dynamics of Salmonella enterica infections.

An understanding of within-host dynamics of pathogen interactions with eukaryotic cells can shape the development of effective preventive measures and drug regimes. Such investigations have been hampered by the difficulty of identifying and observing directly, within live tissues, the multiple key variables that underlay infection processes. Fluorescence microscopy data on intracellular distributions of Salmonella enterica serovar Typhimurium (S. Typhimurium) show that, while the number of infected cells increases with time, the distribution of bacteria between cells is stationary (though highly skewed). Here, we report a simple model framework for the intensity of intracellular infection that links the quasi-stationary distribution of bacteria to bacterial and cellular demography. This enables us to reject the hypothesis that the skewed distribution is generated by intrinsic cellular heterogeneities, and to derive specific predictions on the within-cell dynamics of Salmonella division and host-cell lysis. For within-cell pathogens in general, we show that within-cell dynamics have implications across pathogen dynamics, evolution, and control, and we develop novel generic guidelines for the design of antibacterial combination therapies and the management of antibiotic resistance.

Enhanced virulence of Salmonella enterica serovar typhimurium after passage through mice.

The interaction between Salmonella enterica and the host immune system is complex. The outcome of an infection is the result of a balance between the in vivo environment where the bacteria survive and grow and the regulation of fitness genes at a level sufficient for the bacteria to retain their characteristic rate of growth in a given host. Using bacteriological counts from tissue homogenates and fluorescence microscopy to determine the spread, localization, and distribution of S. enterica in the tissues, we show that, during a systemic infection, S. enterica adapts to the in vivo environment. The adaptation becomes a measurable phenotype when bacteria that have resided in a donor animal are introduced into a recipient naïve animal. This adaptation does not confer increased resistance to early host killing mechanisms but can be detected as an enhancement in the bacterial net growth rate later in the infection. The enhanced growth rate is lost upon a single passage in vitro, and it is therefore transient and not due to selection of mutants. The adapted bacteria on average reach higher intracellular numbers in individual infected cells and therefore have patterns of organ spread different from those of nonadapted bacteria. These experiments help in developing an understanding of the influence of passage in a host on the fitness and virulence of S. enterica.

Intracellular demography and the dynamics of Salmonella enterica infections.

An understanding of within-host dynamics of pathogen interactions with eukaryotic cells can shape the development of effective preventive measures and drug regimes. Such investigations have been hampered by the difficulty of identifying and observing directly, within live tissues, the multiple key variables that underlay infection processes. Fluorescence microscopy data on intracellular distributions of Salmonella enterica serovar Typhimurium (S. Typhimurium) show that, while the number of infected cells increases with time, the distribution of bacteria between cells is stationary (though highly skewed). Here, we report a simple model framework for the intensity of intracellular infection that links the quasi-stationary distribution of bacteria to bacterial and cellular demography. This enables us to reject the hypothesis that the skewed distribution is generated by intrinsic cellular heterogeneities, and to derive specific predictions on the within-cell dynamics of Salmonella division and host-cell lysis. For within-cell pathogens in general, we show that within-cell dynamics have implications across pathogen dynamics, evolution, and control, and we develop novel generic guidelines for the design of antibacterial combination therapies and the management of antibiotic resistance.

In vivo dynamics of the internal fibrous structure in smooth adhesive pads of insects.

Many insects with smooth adhesive pads can rapidly enlarge their contact area by centripetal pulls on the legs, allowing them to cope with sudden mechanical perturbations such as gusts of wind or raindrops. The short time scale of this reaction excludes any neuromuscular control; it is thus more likely to be caused by mechanical properties of the pad's specialized cuticle. This soft cuticle contains numerous branched fibrils oriented almost perpendicularly to the surface. Assuming a fixed volume of the water-filled cuticle, we hypothesized that pulls could decrease the fibril angle, thereby helping the contact area to expand laterally and longitudinally. Three-dimensional fluorescence microscopy on the cuticle of smooth stick insect pads confirmed that pulls significantly reduced the fibril angle. However, the fibril angle variation appeared insufficient to explain the observed increase in contact area. Direct strain measurements in the contact zone demonstrated that pulls not only expand the cuticle laterally, but also add new contact area at the pad's outer edge.

Quantification of the effects of antibodies on the extra- and intracellular dynamics of Salmonella enterica.

Antibodies are known to be essential in controlling Salmonella infection, but their exact role remains elusive. We recently developed an in vitro model to investigate the relative efficiency of four different human immunoglobulin G (IgG) subclasses in modulating the interaction of the bacteria with human phagocytes. Our results indicated that different IgG subclasses affect the efficacy of Salmonella uptake by human phagocytes. In this study, we aim to quantify the effects of IgG on intracellular dynamics of infection by combining distributions of bacterial numbers per phagocyte observed by fluorescence microscopy with a mathematical model that simulates the in vitro dynamics. We then use maximum likelihood to estimate the model parameters and compare them across IgG subclasses. The analysis reveals heterogeneity in the division rates of the bacteria, strongly suggesting that a subpopulation of intracellular Salmonella, while visible under the microscope, is not dividing. Clear differences in the observed distributions among the four IgG subclasses are best explained by variations in phagocytosis and intracellular dynamics. We propose and compare potential factors affecting the replication and death of bacteria within phagocytes, and we discuss these results in the light of recent findings on dormancy of Salmonella.

Scanning gate microscopy of current-annealed single layer graphene

We have used scanning gate microscopy to explore the local conductivity of a current-annealed graphene flake. A map of the local neutrality point (NP) after annealing at low current density exhibits micron-sized inhomogeneities. Broadening of the local e-h transition is also correlated with the inhomogeneity of the NP. Annealing at higher current density reduces the NP inhomogeneity, but we still observe some asymmetry in the e-h conduction. We attribute this to a hole-doped domain close to one of the metal contacts combined with underlying striations in the local NP. © 2010 American Institute of Physics.

Observation of the ordered growth of coronene on Ag(111): a scanning tunnelling microscopy study of vacuum deposited molecules

The molecular ordering of coronene (C24H12) obtained by vacuum-deposition onto predominantly Ag(111) on mica has been investigated using the scanning tunnelling microscope. Real-space topographic images reveal that in certain regions we obtain layer-by-layer ordered growth of the molecules on this substrate which agrees with previous indirect measurements (the growth did not display this ordering in other regions). In our experiments on the ordered regions, we observe the best imaging contrast at a voltage bias of -0.28 V which may correspond to a resonant tunnelling process through the molecules. © 1995.

Scanning transmission electron microscopy investigation of the Si(111)/AlN interface grown by metalorganic vapor phase epitaxy

The structure and chemistry of the interface between a Si(111) substrate and an AlN(0001) thin film grown by metalorganic vapor phase epitaxy have been investigated at a subnanometer scale using high-angle annular dark field imaging and electron energy-loss spectroscopy. 〈1120̄〉AlN ∥ 〈110〉Si and 〈0001〉AlN ∥ 〈111〉 Si epitaxial relations were observed and an Al-face polarity of the AlN thin film was determined. Despite the use of Al deposition on the Si surface prior to the growth, an amorphous interlayer of composition SiNx was identified at the interface. Mechanisms leading to its formation are discussed. © 2010 American Institute of Physics.

Curvature and wrinkling of premixed flame Kernels comparison between planar laser induced fluorescence (PLIF) and 3D direct numerical simulations (DNS)

A direct comparison between time resolved PLIF measurements of OH and two dimensional slices from a full three dimensional DNS data set of turbulent premixed flame kernels in lean methane/air mixture was presented. The local flame structure and the degree of flame wrinkling were examined in response to differing turbulence intensities and turbulent Reynolds numbers. Simulations were performed using the SEGA DNS code, which is based on the solution of the compressible Navier Stokes, species, and energy equations for a lean hydrocarbon mixture. For the OH PLIF measurements, a cluster of four Nd:YAG laser was fired sequentially at high repetition rates and used to pump a dye laser. The frequency doubled laser beam was formed into a sheet of 40 mm height using a cylindrical telescope. The combination of PLIF and DNS has been demonstrated as a powerful tool for flame analysis. This research will form the basis for the development of sub-grid-scale (SGS) models for LES of lean-premixed combustion systems such as gas turbines. This is an abstract of a paper presented at the 30th International Symposium on Combustion (Chicago, IL 7/25-30/2004).

Electron microscopy of polymer-carbon nanotubes composites

Characterization of polymer nanocomposites by electron microscopy has been attempted since last decade. Main drives for this effort were analysis of dispersion and alignment of fillers in the matrix. Sample preparation, imaging modes and irradiation conditions became particularly challenging due to the small dimension of the fillers and also to the mechanical and conductive differences between filler and matrix. To date, no standardized dispersion and alignment process or characterization procedures exist in the trade. Review of current state of the art on characterization of polymer nanocomposites suggests that the most innovative electron and ion beam microscopy has not yet been deployed in this material system. Additionally, recently discovered functionalities of these composites, such as electro and photoactuation are amenable to the investigation of the atomistic phenomena by in situ transmission electron microscopy. The possibility of using innovative thinning techniques is presented. © 2010 Copyright SPIE - The International Society for Optical Engineering.

Osteoblast and monocyte responses to 444 ferritic stainless steel intended for a Magneto-Mechanically Actuated Fibrous Scaffold

The rationale behind this work is to design an implant device, based on a ferromagnetic material, with the potential to deform in vivo promoting osseointegration through the growth of a healthy periprosthetic bone structure. One of the primary requirements for such a device is that the material should be non-inflammatory and non-cytotoxic. In the study described here, we assessed the short-term cellular response to 444 ferritic stainless steel; a steel, with a very low interstitial content and a small amount of strong carbide-forming elements to enhance intergranular corrosion resistance. Two different human cell types were used: (i) foetal osteoblasts and (ii) monocytes. Austenitic stainless steel 316L, currently utilised in many commercially available implant designs, and tissue culture plastic were used as the control surfaces. Cell viability, proliferation and alkaline phosphatase activity were measured. In addition, cells were stained with alizarin red and fluorescently-labelled phalloidin and examined using light, fluorescence and scanning electron microscopy. Results showed that the osteoblast cells exhibited a very similar degree of attachment, growth and osteogenic differentiation on all surfaces. Measurement of lactate dehydrogenase activity and tumour necrosis factor alpha protein released from human monocytes indicated that 444 stainless steel did not cause cytotoxic effects or any significant inflammatory response. Collectively, the results suggest that 444 ferritic stainless steel has the potential to be used in advanced bone implant designs. © 2011 Elsevier Ltd.