Inferring diffusion in single live cells at the single-molecule level

The movement of molecules inside living cells is a fundamental feature of biological processes. The ability to both observe and analyse the details of molecular diffusion in vivo at the single-molecule and single-cell level can add significant insight into understanding molecular architectures of diffus- ing molecules and the nanoscale environment in which the molecules diffuse. The tool of choice for monitoring dynamic molecular localization in live cells is fluorescence microscopy, especially so combining total internal reflection fluorescence with the use of fluorescent protein (FP) reporters in offering exceptional imaging contrast for dynamic processes in the cell mem- brane under relatively physiological conditions compared with competing single-molecule techniques. There exist several different complex modes of diffusion, and discriminating these from each other is challenging at the mol- ecular level owing to underlying stochastic behaviour. Analysis is traditionally performed using mean square displacements of tracked particles; however, this generally requires more data points than is typical for single FP tracks owing to photophysical instability. Presented here is a novel approach allowing robust Bayesian ranking of diffusion processes to dis-criminate multiple complex modes probabilistically. It is a computational approach that biologists can use to understand single-molecule features in live cells.

Interfacial Phenomena in Pharmaceutical Low Moisture Content Powder Processing

Powders are essential materials in the pharmaceutical industry, being involved in majority of all drug manufacturing. Powder flow and particle size are central particle properties addressed by means of particle engineering. The aim of the thesis was to gain knowledge on powder processing with restricted liquid addition, with a primary focus on particle coating and early granule growth. Furthermore, characterisation of this kind of processes was performed. A thin coating layer of hydroxypropyl methylcellulose was applied on individual particles of ibuprofen in a fluidised bed top-spray process. The polymeric coating improved the flow properties of the powder. The improvement was strongly related to relative humidity, which can be seen as an indicator of a change in surface hydrophilicity caused by the coating. The ibuprofen used in the present study had a d50 of 40 μm and thus belongs to the Geldart group C powders, which can be considered as challenging materials in top-spray coating processes. Ibuprofen was similarly coated using a novel ultrasound-assisted coating method. The results were in line with those obtained from powders coated in the fluidised bed process mentioned above. It was found that the ultrasound-assisted method was capable of coating single particles with a simple and robust setup. Granule growth in a fluidised bed process was inhibited by feeding the liquid in pulses. The results showed that the length of the pulsing cycles is of importance, and can be used to adjust granule growth. Moreover, pulsed liquid feed was found to be of greater significance to granule growth in high inlet air relative humidity. Liquid feed pulsing can thus be used as a tool in particle size targeting in fluidised bed processes and in compensating for changes in relative humidity of the inlet air. The nozzle function of a two-fluid external mixing pneumatic nozzle, typical for small scale pharmaceutical fluidised bed processes, was studied in situ in an ongoing fluidised bed process with particle tracking velocimetry. It was found that the liquid droplets undergo coalescence as they proceed away from the nozzle head. The coalescence was expected to increase droplet speed, which was confirmed in the study. The spray turbulence was studied, and the results showed turbulence caused by the event of atomisation and by the oppositely directed fluidising air. It was concluded that particle tracking velocimetry is a suitable tool for in situ spray characterisation. The light transmission through dense particulate systems was found to carry information on particle size and packing density as expected based on the theory of light scattering by solids. It was possible to differentiate binary blends consisting of components with differences in optical properties. Light transmission showed potential as a rapid, simple and inexpensive tool in characterisation of particulate systems giving information on changes in particle systems, which could be utilised in basic process diagnostics.

Spatial Filter Based Bessel-Like Beam for Improved Penetration Depth Imaging in Fluorescence Microscopy

Monitoring and visualizing specimens at a large penetration depth is a challenge. At depths of hundreds of microns, several physical effects (such as, scattering, PSF distortion and noise) deteriorate the image quality and prohibit a detailed study of key biological phenomena. In this study, we use a Bessel-like beam in-conjugation with an orthogonal detection system to achieve depth imaging. A Bessel-like penetrating diffractionless beam is generated by engineering the back-aperture of the excitation objective. The proposed excitation scheme allows continuous scanning by simply translating the detection PSF. This type of imaging system is beneficial for obtaining depth information from any desired specimen layer, including nano-particle tracking in thick tissue. As demonstrated by imaging the fluorescent polymer-tagged-CaCO3 particles and yeast cells in a tissue-like gel-matrix, the system offers a penetration depth that extends up to 650 mu m. This achievement will advance the field of fluorescence imaging and deep nano-particle tracking.

Microscopic dynamics of nanoparticle monolayers at air-water interface

We present results of surface mechanical and particle tracking measurements of nanoparticles trapped at the air-water interface as a function of their areal density. We monitor both the surface pressure (II) and isothermal compression modulus (epsilon) as well as the dynamics of nanoparticle clusters, using fluorescence confocal microscopy while they are compressed to very high density near the two dimensional close packing density Phi similar to 0.82. We observe non-monotonic variation in both epsilon and the dynamic heterogeneity, characterized by the dynamical susceptibility chi(4) with Phi, in such high density monolayers. We provide insight into the underlying nature of such transitions in close packed high density nanoparticle monolayers in terms of the morphology and flexibility of these soft colloidal particles.. We discuss the significance our results in the context of related studies on two dimensional granular or colloidal systems. (C) 2013 Elsevier Inc. All rights reserved.

Life of flame particles embedded in premixed flames interacting with near isotropic turbulence

Flame particles are surface points that always remain embedded on, by comoving with a given iso-scalar surface within a flame. Tracking flame particles allow us to study the fate of propagating surface locations uniquely identified throughout their evolution with time. In this work, using Direct Numerical Simulations we study the finite lifetime of such flame particles residing on iso-temperature surfaces of statistically planar H-2-air flames interacting with near-isotropic turbulence. We find that individual flame particles as well as their ensemble, experience progressively increasing tangential straining rate (K-t) and increasing negative curvature (kappa) near the end of their lifetime to finally get annihilated. By studying two different turbulent flow conditions, flame particle tracking shows that such tendency of local flame surfaces to be strained and cusped towards pinch-off from the main surface is a rather generic feature, independent of initial conditions, locations and ambient turbulence intensity levels. The evolution of the alignments between the flame surface normals and the principal components of the local straining rates are also tracked. We find that the surface normals initially aligned with the most extensive principal strain rate components, rotate near the end of flame particles' lifetime to enable preferential alignment between the surface tangent and the most extensive principal strain rate component. This could explain the persistently increasing tangential strain rate, sharp negative curvature formation and eventual detachment. (C) 2014 The Combustion Institute. Published by Elsevier Inc. All rights reserved.

Airblast spray in crossflow - Structure, trajectory and droplet sizing

This study reports results of an experimental investigation of airblast spray of water and ethanol in crossflow. Laser shadowgraphy and Particle/Droplet Imaging Analysis (PDIA) are used to derive spray trajectory and drop size information while Particle Tracking Velocimetry (PTV) is used to measure droplet velocities. A new phenomenon of spray bifurcation is observed for low Gas to Liquid Ratio (GLR) cases. The reasons for the spatial bifurcation can be attributed to a combination of reasons. These are (a) presence of large ligaments and droplets in the near-nozzle region for low GLRs (b) secondary breakup experienced by ligaments/droplets leading to formation of a large number of small droplets, and (c) the crossflow causing differential dispersion of the small and large droplets. A novel correlation for spray trajectory is proposed incorporating the momentum ratio and liquid surface tension. This correlation is shown to be effective in predicting the non-linear spray trajectory over a large range of conditions for not only water but ethanol and Jet-A also. It is observed that the larger droplets penetrate further into the crossflow, in the direction of injection. Thus, with increase in height of the measurement location from the injection plane, the droplet Sauter Mean Diameter (SMD) is found to increase. Moreover, as the droplets travel downstream in the crossflow direction, the droplet SMD is observed to decrease. The effect of drag is assessed by comparing velocity of different sizes of droplets at various locations. Smaller droplets are entrained into the crossflow at much lower elevations, whereas larger droplets tend to penetrate further into the crossflow. (C) 2015 Elsevier Ltd. All rights reserved.

TURBULENCE-TRANSPORT-CHEMISTRY INTERACTION IN STATISTICALLY PLANAR PREMIXED FLAMES AND IGNITION KERNELS IN NEAR ISOTROPIC TURBULENCE

Turbulence-transport-chemistry interaction plays a crucial role on the flame surface geometry, local and global reactionrates, and therefore, on the propagation and extinction characteristics of intensely turbulent, premixed flames encountered in LPP gas-turbine combustors. The aim of the present work is to understand these interaction effects on the flame surface annihilation and extinction of lean premixed flames, interacting with near isotropic turbulence. As an example case, lean premixed H-2-air mixture is considered so as to enable inclusion of detailed chemistry effects in Direct Numerical Simulations (DNS). The work is carried out in two phases namely, statistically planar flames and ignition kernel, both interacting with near isotropic turbulence, using the recently proposed Flame Particle Tracking (FPT) technique. Flame particles are surface points residing and commoving with an iso-scalar surface within a premixed flame. Tracking flame particles allows us to study the evolution of propagating surface locations uniquely identified with time. In this work, using DNS and FPT we study the flame speed, reaction rate and transport histories of such flame particles residing on iso-scalar surfaces. An analytical expression for the local displacement flame speed (SO is derived, and the contribution of transport and chemistry on the displacement flame speed is identified. An examination of the results of the planar case leads to a conclusion that the cause of variation in S-d may be attributed to the effects of turbulent transport and heat release rate. In the second phase of this work, the sustenance of an ignition kernel is examined in light of the S-curve. A newly proposed Damkohler number accounting for local turbulent transport and reaction rates is found to explain either the sustenance or otherwise propagation of flame kernels in near isotropic turbulence.

Kinematic flow patterns in slow deformation of a dense granular material

The kinematic flow pattern in slow deformation of a model dense granular medium is studied at high resolution using in situ imaging, coupled with particle tracking. The deformation configuration is indentation by a flat punch under macroscopic plane-strain conditions. Using a general analysis method, velocity gradients and deformation fields are obtained from the disordered grain arrangement, enabling flow characteristics to be quantified. The key observations are the formation of a stagnation zone, as in dilute granular flow past obstacles; occurrence of vortices in the flow immediately underneath the punch; and formation of distinct shear bands adjoining the stagnation zone. The transient and steady state stagnation zone geometry, as well as the strength of the vortices and strain rates in the shear bands, are obtained from the experimental data. All of these results are well-reproduced in exact-scale non-smooth contact dynamics simulations. Full 3D numerical particle positions from the simulations allow extraction of flow features that are extremely difficult to obtain from experiments. Three examples of these, namely material free surface evolution, deformation of a grain column below the punch and resolution of velocities inside the primary shear band, are highlighted. The variety of flow features observed in this model problem also illustrates the difficulty involved in formulating a complete micromechanical analytical description of the deformation.

Discussions on slip length measurements by microPIV/PTV in microchannels

Fluid transportation in microfluidic system could be benefit from the slip on solid-liquid interface. Slip length on many kinds of hydrophilic/hydrophobic surfaces have been measured recently. The two common-used experimental methods for boundary slip measurement include: (1) surface force measurement, such as surface force apparatus (SFA), atom force microscope (AFM), and (2) velocity measurement, like microPIV/PTV (Particle image velocimetry / Particle tracking velocimetry), total internal reflection velocimetry (TIRV). However, the measured results are rather scattered, larger measured slip lengths were reported by microPIV/PTV experiments. In this paper, we will investigate the deviations of the measured slip length on smooth hydrophilic surface. After measuring detailed velocity profiles very close to hydrophilic glass wall, we give a discussion on the effects influencing the slip measurements.

Direct visualization of the dynamics of membrane-anchor proteins in living cells

Dynamic properties of proteins have crucial roles in understanding protein function and molecular mechanism within cells. In this paper, we combined total internal reflection fluorescence microscopy with oblique illumination fluorescence microscopy to observe directly the movement and localization of membrane-anchored green fluorescence proteins in living cells. Total internal reflect illumination allowed the observation of proteins in the cell membrane of living cells since the penetrate depth could be adjusted to about 80 nm, and oblique illumination allowed the observation of proteins both in the cytoplasm and apical membrane, which made this combination a promising tool to investigate the dynamics of proteins through the whole cell. Not only individual protein molecule tracks have been analyzed quantitatively but also cumulative probability distribution function analysis of ensemble trajectories has been done to reveal the mobility of proteins. Finally, single particle tracking has acted as a compensation for single molecule tracking. All the results exhibited green fluorescence protein dynamics within cytoplasm, on the membrane and from cytoplasm to plasma membrane.

Investigation of air-core vortex at hydraulic intakes

Hydrodynamic properties of the surface vortex have been investigated. Based on the Navier-Stokes equations, three sets of the new formulations for the tangential velocity distributions are derived, and verified against the experimental measurements in the literature. It is shown that one modification greatly improves the agreement with the experimental data. Physical model experiments were carried out to study the intake vortex related to the Xiluodu hydropower project. The velocity fields were measured using the Particle Tracking Velocimetry (PTV) technique. The proposed equation for tangential velocity distribution is applied to the Xiluodu project with the solid boundary being considered by the method of images. Good agreement has been observed between the formula prediction and the experimental observation. © 2010 Publishing House for Journal of Hydrodynamics.

Differential Dynamic Microscopy: a High-Throughput Method for Characterizing the Motility of Microorganism

We present a fast, high-throughput method for characterizing the motility of microorganisms in 3D based on standard imaging microscopy. Instead of tracking individual cells, we analyse the spatio-temporal fluctuations of the intensity in the sample from time-lapse images and obtain the intermediate scattering function (ISF) of the system. We demonstrate our method on two different types of microorganisms: bacteria, both smooth swimming (run only) and wild type (run and tumble) Escherichia coli, and the bi-flagellate alga Chlamydomonas reinhardtii. We validate the methodology using computer simulations and particle tracking. From the ISF, we are able to extract (i) for E. coli: the swimming speed distribution, the fraction of motile cells and the diffusivity, and (ii) for C. reinhardtii: the swimming speed distribution, the amplitude and frequency of the oscillatory dynamics. In both cases, the motility parameters are averaged over \approx 10^4 cells and obtained in a few minutes.

基于栅格地图的多化学羽流源定位

本文研究了移动机器人在湍流环境中定位多个化学羽流源的问题。利用粒子随机行走方法建立动态羽流模型。在此环境中机器人进行梳状搜索并采集羽流数据,使用一种基于后验概率独立假设的改进贝叶斯算法融合这些传感器数据建立一张羽流源位置的概率地图,地图中具有高概率值的栅格指出了羽流源可能的位置。仿真结果说明了该方法的有效性,通过与标准贝叶斯算法的比较说明了该方法优点。

Three-phase computations of the continuous casting process

In this paper, the continuous casting process for steel slab production is modelled using a mult-physics approach. For this purpose, a Finite Volume (FV) numerical model was constructed in 3D, with the following characteristics: Time dependent, turbulent fluid flow and heat transfer in the molten steel and flux regions, solidification of the skin layer, under prescribed heat loss boundary conditions, particle tracking simulation of argon bubbles injected with the metal into the mould, full coupling between bubbles and liquid through buoyancy and interfacial forces using a novel gas accumulation technique, and a full transient simulation of flux-metal interface behaviour under the influence of gravity and fluid inertial forces and bubble plume buoyancy. The unstructure mesh FV code PHYSICA developed at Greenwich was used for carry out the simulations with physical process data and properties supplied by IRSID SA.

Dynamic model for metal cleanness evaluation by melting in a cold crucible

Melting of metallic samples in a cold crucible causes inclusions to concentrate on the surface owing to the action of the electromagnetic force in the skin layer. This process is dynamic, involving the melting stage, then quasi-stationary particle separation, and finally the solidification in the cold crucible. The proposed modeling technique is based on the pseudospectral solution method for coupled turbulent fluid flow, thermal and electromagnetic fields within the time varying fluid volume contained by the free surface, and partially the solid crucible wall. The model uses two methods for particle tracking: (1) a direct Lagrangian particle path computation and (2) a drifting concentration model. Lagrangian tracking is implemented for arbitrary unsteady flow. A specific numerical time integration scheme is implemented using implicit advancement that permits relatively large time-steps in the Lagrangian model. The drifting concentration model is based on a local equilibrium drift velocity assumption. Both methods are compared and demonstrated to give qualitatively similar results for stationary flow situations. The particular results presented are obtained for iron alloys. Small size particles of the order of 1 μm are shown to be less prone to separation by electromagnetic field action. In contrast, larger particles, 10 to 100 μm, are easily “trapped” by the electromagnetic field and stay on the sample surface at predetermined locations depending on their size and properties. The model allows optimization for melting power, geometry, and solidification rate.