M. R. Brown

Single cell nanoparticle tracking to model cell cycle dynamics and compartmental inheritance

Single cell encoding with quantum dots as live cell optical tracers for deriving proliferation parameters has been developed using modelling to investigate cell cycle and proliferative outputs of human osteosarcoma cells undergoing mitotic bypass and endocycle routing. A computer-based simulation of the evolving cell population provides information on the dilution and segregation of nanoparticle dose cell by cell division and allows quantitative assessment of patterns of division, at both single cell and including whole population level cell cycle routing, with no a-priori knowledge of the population proliferation potential. The output therefore provides a unique mitotic distribution function that represents a convolution of cell cycle kinetics (cell division) and the partitioning coefficient for the labelled cell compartment (daughter-daughter inheritance or lineage asymmetry). The current study has shown that the cellular quantum dot fluorescence reduced over time as the particles were diluted by the process of cell division and had the properties of a non-random highly asymmetric event. Asymmetric nanoparticle segregation in the endosomal compartment has major implications on cell-fate determining signaling pathways and could lead to an understanding of the origins of unique proliferation and drug-resistance characteristics within a tumour cell lineage.

Quantification of Nanoparticle Dose and Vesicular Inheritance in Proliferating Cells

Assessing dose in nanoparticle–cell interactions is inherently difficult due to a complex multiplicity of possible mechanisms and metrics controlling particle uptake. The fundamental unit of nanoparticle dose is the number of particles internalized per cell; we show that this can be obtained for large cell populations that internalize fluorescent nanoparticles by endocytosis, through calibration of cytometry measurements to transmission electron microscopy data. Low-throughput, high-resolution electron imaging of quantum dots in U-2 OS cells is quantified and correlated with high-throughput, low-resolution optical imaging of the nanoparticle-loaded cells. From the correlated data, we obtain probability distribution functions of vesicles per cell and nanoparticles per vesicle. Sampling of these distributions and comparison to fluorescence intensity histograms from flow cytometry provide the calibration factor required to transform the cytometry metric to total particle dose per cell, the mean value of which is 2.4 million. Use of the probability distribution functions to analyze particle partitioning during cell division indicates that, while vesicle inheritance is near symmetric, highly variable vesicle loading leads to a highly asymmetric particle dose within the daughter cells.

Modeling multiple quantum barrier effects and reduced electron leakage in red-emitting laser diodes

Severe electron leakage impedes the full exploitation of AlGaInP laser diodes in the 630nm regime. Such thermally activated currents are attributed to inherently small conduction band offsets and intervalley transfer between the Γ and X conduction band minima. To negate the detrimental effect of these two intrinsic material issues a theoretical model is proposed. A multi-quantum-barrier (MQB) structure able to inhibit both Γ- and X-band transmissions is inserted in the p-doped region adjacent to the active region of the device, allowing a greater percentage of injected electrons to be reflected back within the active region. The design of the MQB follows a strict optimization procedure that takes into account fluctuations of superlattice layer width and composition. This model is used in conjunction with a dual conduction band drift-diffusion simulator to enable the design of the MQB at an operating voltage and hence account for nonlinear charge distribution across it. Initial results indicate strong agre...

Fractal dimension (df) as a new structural biomarker of clot microstructure in different stages of lung cancer

Venous thromboembolism (VTE) is common in cancer patients, and is the second commonest cause of death associated with the disease. Patients with chronic inflammation, such as cancer, have been shown to have pathological clot structures with modulated mechanical properties. Fractal dimension (df) is a new technique which has been shown to act as a marker of the microstructure and mechanical properties of blood clots, and can be performed more readily than current methods such as scanning electron microscopy (SEM). We measured df in 87 consecutive patients with newly diagnosed lung cancer prior to treatment and 47 matched-controls. Mean group values were compared for all patients with lung cancer vs controls and for limited disease vs extensive disease. Results were compared with conventional markers of coagulation, fibrinolysis and SEM images. Significantly higher values of df were observed in lung cancer patients compared with controls and patients with extensive disease had higher values than those with limited disease (p< 0.05), whilst conventional markers failed to distinguish between these groups. The relationship between df of the incipient clot and mature clot microstructure was confirmed by SEM and computational modelling: higher df was associated with highly dense clots formed of smaller fibrin fibres in lung cancer patients compared to controls. This study demonstrates that df is a sensitive technique which quantifies the structure and mechanical properties of blood clots in patients with lung cancer. Our data suggests that df has the potential to identify patients with an abnormal clot microstructure and greatest VTE risk.

Automated cell identification and tracking using nanoparticle moving-light-displays.

An automated technique for the identification, tracking and analysis of biological cells is presented. It is based on the use of nanoparticles, enclosed within intra-cellular vesicles, to produce clusters of discrete, point-like fluorescent, light sources within the cells. Computational analysis of these light ensembles in successive time frames of a movie sequence, using k-means clustering and particle tracking algorithms, provides robust and automated discrimination of live cells and their motion and a quantitative measure of their proliferation. This approach is a cytometric version of the moving light display technique which is widely used for analyzing the biological motion of humans and animals. We use the endocytosis of CdTe/ZnS, core-shell quantum dots to produce the light displays within an A549, epithelial, lung cancer cell line, using time-lapse imaging with frame acquisition every 5 minutes over a 40 hour time period. The nanoparticle moving light displays provide simultaneous collection of cell motility data, resolution of mitotic traversal dynamics and identification of familial relationships allowing construction of multi-parameter lineage trees.

The effect of interface roughness on multilayer heterostructures

Semiconductor devices which utilize the quantum confinement of charge carriers inherently employ material layers thin enough that even monolayer interface roughness has an effect on performance. We present a method for including the effect of interface roughness on the calculation of electron energy levels and wavefunctions by solving Schrodinger’s equation across the interface between semiconductor layers. Interface roughness is approximated by considering a supplementary interface in addition to the idealized perfectly flat interface. The position of the second interface is considered to be a probabilistic distribution with a mean corresponding to the position of the perfect case. Using Green’s theorem and the appropriate reciprocity relations, we deduce a correction to the reflection and transmission probabilities of an electron incident upon a rough material interface. The procedure is presented in terms of a transfer matrix algorithm to facilitate use in existing electron reflection transmission prob...

Effects of unidirectional flow shear stresses on the formation, fractal microstructure and rigidity of incipient whole blood clots and fibrin gels

Abstract Incipient clot formation in whole blood and fibrin gels was studied by the rheometric techniques of controlled stress parallel superposition (CSPS) and small amplitude oscillatory shear (SAOS). The effects of unidirectional shear stress on incipient clot microstructure, formation kinetics and elasticity are reported in terms of the fractal dimension (df) of the fibrin network, the gel network formation time (TGP) and the shear elastic modulus, respectively. The results of this first haemorheological application of CSPS reveal the marked sensitivity of incipient clot microstructure to physiologically relevant levels of shear stress, these being an order of magnitude lower than have previously been studied by SAOS. CSPS tests revealed that exposure of forming clots to increasing levels of shear stress produces a corresponding elevation in df, consistent with the formation of tighter, more compact clot microstructures under unidirectional flow. A corresponding increase in shear elasticity was recorded. The scaling relationship established between shear elasticity and df for fibrin clots and whole blood confirms the fibrin network as the dominant microstructural component of the incipient clot in terms of its response to imposed stress. Supplementary studies of fibrin clot formation by rheometry and microscopy revealed the substantial additional network mass required to increase df and provide evidence to support the hypothesis that microstructural changes in blood clotted under unidirectional shear may be attributed to flow enhanced thrombin generation and activation. CSPS also identified a threshold value of unidirectional shear stress above which no incipient clot formation could be detected. CSPS was shown to be a valuable haemorheological tool for the study of the effects of physiological and pathological levels of shear on clot properties.

Direct real-time observation of catastrophic optical degradation in operating semiconductor lasers using scanning tunneling microscopy

Cross-sectional scanning tunneling microscopy is performed on operating semiconductor quantum well laser devices to reveal real time changes in device structure. Low and nominally doped capping regions adjacent to the quantum well active region are found to heat under normal operating conditions. The increase in anion-vacancy defect formation and the generation of surface states pins the Fermi level at the surface and begins the process of catastrophic optical degradation which eventually destroys the device. The technique has implications for the study of defect generation and in-operation changes in all nanostructures.

An investigation of multi-quantum barriers for band offset engineering in AlGaInP/GaInP lasers

In this paper, we present a critical study of a multi-quantum barrier (MQB) structure fabricated using the Al0.5In0.5P/Ga0.5In0.5P material system. The structure was optimised theoretically based on a single Γ band model. When an identical structure was placed in a quantum well laser device, no improvement in the threshold current was observed compared to QW laser structure without an MQB. Cross-sectional scanning tunnelling microscopy (STM) was used to assess the structural quality of the MQB structure for the first time. Factors affecting the limited barrier enhancement are discussed in terms of the interfacial quality of the Al0.5In0.5P/Ga0.5In0.5P interface in the superlattice of the MQB and the need to consider multi-valley transport. The paper highlights some of the fundamental problems that must be overcome for MQBs to be a viable method to improve red laser efficiency.

European sea bass show behavioural resilience to near-future ocean acidification.

Ocean acidification (OA)—caused by rising concentrations of carbon dioxide (CO2)—is thought to be a major threat to marine ecosystems and has been shown to induce behavioural alterations in fish. Here we show behavioural resilience to near-future OA in a commercially important and migratory marine finfish, the Sea bass (Dicentrarchus labrax). Sea bass were raised from eggs at 19°C in ambient or near-future OA (1000 µatm pCO2) conditions and n = 270 fish were observed 59–68 days post-hatch using automated tracking from video. Fish reared under ambient conditions, OA conditions, and fish reared in ambient conditions but tested in OA water showed statistically similar movement patterns, and reacted to their environment and interacted with each other in comparable ways. Thus our findings indicate behavioural resilience to near-future OA in juvenile sea bass. Moreover, simulated agent-based models indicate that our analysis methods are sensitive to subtle changes in fish behaviour. It is now important to determine whether the absences of any differences persist under more ecologically relevant circumstances and in contexts which have a more direct bearing on individual fitness.