Cell invasion with proliferation mechanisms motivated by time-lapse data

Cell invasion involves a population of cells which are motile and proliferative. Traditional discrete models of proliferation involve agents depositing daughter agents on nearest- neighbor lattice sites. Motivated by time-lapse images of cell invasion, we propose and analyze two new discrete proliferation models in the context of an exclusion process with an undirected motility mechanism. These discrete models are related to a family of reaction- diffusion equations and can be used to make predictions over a range of scales appropriate for interpreting experimental data. The new proliferation mechanisms are biologically relevant and mathematically convenient as the continuum-discrete relationship is more robust for the new proliferation mechanisms relative to traditional approaches.

Hydrological consequences of land-cover change: Quantifying the influence of plants on soil moisture with time-lapse electrical resistivity

Electrical resistivity of soils and sediments is strongly influenced by the presence of interstitial water. Taking advantage of this dependency, electrical-resistivity imaging (ERI) can be effectively utilized to estimate subsurface soil-moisture distributions. The ability to obtain spatially extensive data combined with time-lapse measurements provides further opportunities to understand links between land use and climate processes. In natural settings, spatial and temporal changes in temperature and porewater salinity influence the relationship between soil moisture and electrical resistivity. Apart from environmental factors, technical, theoretical, and methodological ambiguities may also interfere with accurate estimation of soil moisture from ERI data. We have examined several of these complicating factors using data from a two-year study at a forest-grassland ecotone, a boundary between neighboring but different plant communities.At this site, temperature variability accounts for approximately 20-45 of resistivity changes from cold winter to warm summer months. Temporal changes in groundwater conductivity (mean=650 S/cm =57.7) and a roughly 100-S/cm spatial difference between the forest and grassland had only a minor influence on the moisture estimates. Significant seasonal fluctuations in temperature and precipitation had negligible influence on the basic measurement errors in data sets. Extracting accurate temporal changes from ERI can be hindered by nonuniqueness of the inversion process and uncertainties related to time-lapse inversion schemes. The accuracy of soil moisture obtained from ERI depends on all of these factors, in addition to empirical parameters that define the petrophysical soil-moisture/resistivity relationship. Many of the complicating factors and modifying variables to accurately quantify soil moisture changes with ERI can be accounted for using field and theoretical principles.

Wide Field-of-view Microscopes and Endoscopes for Time-lapse Imaging and High-throughput Screening

Wide field-of-view (FOV) microscopy is of high importance to biological research and clinical diagnosis where a high-throughput screening of samples is needed. This thesis presents the development of several novel wide FOV imaging technologies and demonstrates their capabilities in longitudinal imaging of living organisms, on the scale of viral plaques to live cells and tissues.

The ePetri Dish is a wide FOV on-chip bright-field microscope. Here we applied an ePetri platform for plaque analysis of murine norovirus 1 (MNV-1). The ePetri offers the ability to dynamically track plaques at the individual cell death event level over a wide FOV of 6 mm × 4 mm at 30 min intervals. A density-based clustering algorithm is used to analyze the spatial-temporal distribution of cell death events to identify plaques at their earliest stages. We also demonstrate the capabilities of the ePetri in viral titer count and dynamically monitoring plaque formation, growth, and the influence of antiviral drugs.

We developed another wide FOV imaging technique, the Talbot microscope, for the fluorescence imaging of live cells. The Talbot microscope takes advantage of the Talbot effect and can generate a focal spot array to scan the fluorescence samples directly on-chip. It has a resolution of 1.2 μm and a FOV of ~13 mm². We further upgraded the Talbot microscope for the long-term time-lapse fluorescence imaging of live cell cultures, and analyzed the cells’ dynamic response to an anticancer drug.

We present two wide FOV endoscopes for tissue imaging, named the AnCam and the PanCam. The AnCam is based on the contact image sensor (CIS) technology, and can scan the whole anal canal within 10 seconds with a resolution of 89 μm, a maximum FOV of 100 mm × 120 mm, and a depth-of-field (DOF) of 0.65 mm. We also demonstrate the performance of the AnCam in whole anal canal imaging in both animal models and real patients. In addition to this, the PanCam is based on a smartphone platform integrated with a panoramic annular lens (PAL), and can capture a FOV of 18 mm × 120 mm in a single shot with a resolution of 100─140 μm. In this work we demonstrate the PanCam’s performance in imaging a stained tissue sample.

Measuring fast gene dynamics in single cells with time-lapse luminescence microscopy.

Time-lapse fluorescence microscopy is an important tool for measuring in vivo gene dynamics in single cells. However, fluorescent proteins are limited by slow chromophore maturation times and the cellular autofluorescence or phototoxicity that arises from light excitation. An alternative is luciferase, an enzyme that emits photons and is active upon folding. The photon flux per luciferase is significantly lower than that for fluorescent proteins. Thus time-lapse luminescence microscopy has been successfully used to track gene dynamics only in larger organisms and for slower processes, for which more total photons can be collected in one exposure. Here we tested green, yellow, and red beetle luciferases and optimized substrate conditions for in vivo luminescence. By combining time-lapse luminescence microscopy with a microfluidic device, we tracked the dynamics of cell cycle genes in single yeast with subminute exposure times over many generations. Our method was faster and in cells with much smaller volumes than previous work. Fluorescence of an optimized reporter (Venus) lagged luminescence by 15-20 min, which is consistent with its known rate of chromophore maturation in yeast. Our work demonstrates that luciferases are better than fluorescent proteins at faithfully tracking the underlying gene expression.

Time-lapse monitoring of climate effects on earthworks using surface waves

The UK’s transportation network is supported by critical geotechnical assets (cuttings/embankments/dams) that require sustainable, cost-effective management, while maintaining an appropriate service level to meet social, economic, and environmental needs. Recent effects of extreme weather on these geotechnical assets have highlighted their vulnerability to climate variations. We have assessed the potential of surface wave data to portray the climate-related variations in mechanical properties of a clay-filled railway embankment. Seismic data were acquired bimonthly from July 2013 to November 2014 along the crest of a heritage railway embankment in southwest England. For each acquisition, the collected data were first processed to obtain a set of Rayleigh-wave dispersion and attenuation curves, referenced to the same spatial locations. These data were then analyzed to identify a coherent trend in their spatial and temporal variability. The relevance of the observed temporal variations was also verified with respect to the experimental data uncertainties. Finally, the surface wave dispersion data sets were inverted to reconstruct a time-lapse model of S-wave velocity for the embankment structure, using a least-squares laterally constrained inversion scheme. A key point of the inversion process was constituted by the estimation of a suitable initial model and the selection of adequate levels of spatial regularization. The initial model and the strength of spatial smoothing were then kept constant throughout the processing of all available data sets to ensure homogeneity of the procedure and comparability among the obtained VS sections. A continuous and coherent temporal pattern of surface wave data, and consequently of the reconstructed VS models, was identified. This pattern is related to the seasonal distribution of precipitation and soil water content measured on site.

Time-lapse monitoring of fluid induced geophysical property variations within an unstable earthwork using P-wave refraction

A significant portion of the UK’s transportation system relies on a network of geotechnical earthworks (cuttings and embankments) that were constructed more than 100 years ago, whose stability is affected by the change in precipitation patterns experienced over the past few decades. The vulnerability of these structures requires a reliable, cost- and time-effective monitoring of their geomechanical condition. We have assessed the potential application of P-wave refraction for tracking the seasonal variations of seismic properties within an aged clay-filled railway embankment, located in southwest England. Seismic data were acquired repeatedly along the crest of the earthwork at regular time intervals, for a total period of 16 months. P-wave first-break times were picked from all available recorded traces, to obtain a set of hodocrones referenced to the same spatial locations, for various dates along the surveyed period of time. Traveltimes extracted from each acquisition were then compared to track the pattern of their temporal variability. The relevance of such variations over time was compared with the data experimental uncertainty. The multiple set of hodocrones was subsequently inverted using a tomographic approach, to retrieve a time-lapse model of VPVP for the embankment structure. To directly compare the reconstructed VPVP sections, identical initial models and spatial regularization were used for the inversion of all available data sets. A consistent temporal trend for P-wave traveltimes, and consequently for the reconstructed VPVP models, was identified. This pattern could be related to the seasonal distribution of precipitation and soil-water content measured on site.

Time-lapse Monitoring of the Slopes of a Heritage Earthwork by Means of Near-surface Seismic Techniques

A significant portion of UK’s infrastructures earthworks was built more than 100 years ago, without modern construction standards: poor maintenance and the change of precipitations pattern experienced in the past decades are currently compromising their stability, leading to an increasing number of failures. To address the need for a reliable and time-efficient monitoring of earthworks at risk of failure we propose here the use of two established seismic techniques for the characterization of the near surface, MASW and P-wave refraction. We have regularly collected MASW and P-wave refraction data, from March 2014 to February 2015, along 4 reduced-scale seismic lines located on the flanks of a heritage railway embankment located in Broadway, SW of England. We have observed a definite temporal variability in terms of phase velocities of SW dispersion curves and of P-wave travel times. The accurate choice of ad-hoc inversion strategies has allowed to reconstruct reliable VP and VS models through which it is potentially possible to track the temporal variations of geo-mechanical properties of the embankment slopes. The variability over time of seismic data and seismic velocities seems to correlate well with rainfall data recorded in the days immediately preceding the date of acquisition.

Time-lapse geophysical investigations over known archaeological features using electrical resistivity imaging and earth resistance

Electrical methods of geophysical survey are known to produce results that are hard to predict at different times of the year, and under differing weather conditions. This is a problem which can lead to misinterpretation of archaeological features under investigation. The dynamic relationship between a ‘natural’ soil matrix and an archaeological feature is a complex one, which greatly affects the success of the feature’s detection when using active electrical methods of geophysical survey. This study has monitored the gradual variation of measured resistivity over a selection of study areas. By targeting difficult to find, and often ‘missing’ electrical anomalies of known archaeological features, this study has increased the understanding of both the detection and interpretation capabilities of such geophysical surveys. A 16 month time-lapse study over 4 archaeological features has taken place to investigate the aforementioned detection problem across different soils and environments. In addition to the commonly used Twin-Probe earth resistance survey, electrical resistivity imaging (ERI) and quadrature electro-magnetic induction (EMI) were also utilised to explore the problem. Statistical analyses have provided a novel interpretation, which has yielded new insights into how the detection of archaeological features is influenced by the relationship between the target feature and the surrounding ‘natural’ soils. The study has highlighted both the complexity and previous misconceptions around the predictability of the electrical methods. The analysis has confirmed that each site provides an individual and nuanced situation, the variation clearly relating to the composition of the soils (particularly pore size) and the local weather history. The wide range of reasons behind survey success at each specific study site has been revealed. The outcomes have shown that a simplistic model of seasonality is not universally applicable to the electrical detection of archaeological features. This has led to the development of a method for quantifying survey success, enabling a deeper understanding of the unique way in which each site is affected by the interaction of local environmental and geological conditions.

Effect of Sodium Ascorbate and the Time Lapse before Cementation after Internal Bleaching on Bond Strength between Dentin and Ceramic

Purpose: To evaluate the effects of the elapsed time (ET) after nonvital bleaching (NVB) and sodium ascorbate application (10%) (SAA) on the shear bond strength of dentin to ceramic.Materials and Methods: Bovine incisors were selected, internally bleached (35% carbamide peroxide) for 9 days and submitted to the following treatments (n = 10): G1, G2, G3-luting after 1, 7, and 14 days; G4, G5, and G6-luting after SAA, 1, 7, and 14 days, respectively. G7 and G8 were not bleached: G7-luting 24 hours after access cavity sealing; G8-luting 24 hours after access cavity sealing after SAA. After NVB, the vestibular dentin was exposed and flattened. The SAA was applied to the dentin (G4, G5, G6, G8) for 10 minutes, and it was then washed and dried. The dentin was etched (37% phosphoric acid), and an adhesive system (Single Bond 2) was applied. Feldspathic ceramic discs (VM7; 4-mm diameter, 3-mm thick) were luted with a dual-resin agent (RelyX ARC, 3M ESPE Dental Products, St. Paul, MN). After 24 hours, specimens were submitted to shear test on a universal testing machine. The data (MPa) were submitted to ANOVA and Dunnet's test (5%).Results: The means (+/- SD) obtained were (MPa): G1 (14 +/- 4.5), G2 (14.6 +/- 3.1), G3 (14 +/- 3.7), G4 (15.5 +/- 4.6), G5 (19.87 +/- 4.5), G6 (16.5 +/- 3.7), G7 (22.8 +/- 6.2), and G8 (18.9 +/- 5.4). SAA had a significant effect on bond strength (p = 0.0054). The effect of ET was not significant (p = 0.1519). G5 and G6 presented higher values than the other bleached groups (p < 0.05) and similar to G7 and G8 (p > 0.05).Conclusions: After NVB, adhesive luting to dentin is recommended after 7 days if sodium ascorbate has been applied prior to dentin hybridization.

Time-lapse microscopy and classification of 2D human mesenchymal stem cells based on cell shape picks up myogenic from osteogenic and adipogenic differentiation

Current methods to characterize mesenchymal stem cells (MSCs) are limited to CD marker expression, plastic adherence and their ability to differentiate into adipogenic, osteogenic and chondrogenic precursors. It seems evident that stem cells undergoing differentiation should differ in many aspects, such as morphology and possibly also behaviour; however, such a correlation has not yet been exploited for fate prediction of MSCs. Primary human MSCs from bone marrow were expanded and pelleted to form high-density cultures and were then randomly divided into four groups to differentiate into adipogenic, osteogenic chondrogenic and myogenic progenitor cells. The cells were expanded as heterogeneous and tracked with time-lapse microscopy to record cell shape, using phase-contrast microscopy. The cells were segmented using a custom-made image-processing pipeline. Seven morphological features were extracted for each of the segmented cells. Statistical analysis was performed on the seven-dimensional feature vectors, using a tree-like classification method. Differentiation of cells was monitored with key marker genes and histology. Cells in differentiation media were expressing the key genes for each of the three pathways after 21 days, i.e. adipogenic, osteogenic and chondrogenic, which was also confirmed by histological staining. Time-lapse microscopy data were obtained and contained new evidence that two cell shape features, eccentricity and filopodia (= 'fingers') are highly informative to classify myogenic differentiation from all others. However, no robust classifiers could be identified for the other cell differentiation paths. The results suggest that non-invasive automated time-lapse microscopy could potentially be used to predict the stem cell fate of hMSCs for clinical application, based on morphology for earlier time-points. The classification is challenged by cell density, proliferation and possible unknown donor-specific factors, which affect the performance of morphology-based approaches. Copyright © 2012 John Wiley & Sons, Ltd.

TIME LAPSE SEISMIC OBSERVATIONS AND EFFECTS OF RESERVOIR COMPRESSIBILITY AT TEAL SOUTH OIL FIELD

One of the original ocean-bottom time-lapse seismic studies was performed at the Teal South oil field in the Gulf of Mexico during the late 1990’s. This work reexamines some aspects of previous work using modern analysis techniques to provide improved quantitative interpretations. Using three-dimensional volume visualization of legacy data and the two phases of post-production time-lapse data, I provide additional insight into the fluid migration pathways and the pressure communication between different reservoirs, separated by faults. This work supports a conclusion from previous studies that production from one reservoir caused regional pressure decline that in turn resulted in liberation of gas from multiple surrounding unproduced reservoirs. I also provide an explanation for unusual time-lapse changes in amplitude-versus-offset (AVO) data related to the compaction of the producing reservoir which, in turn, changed an isotropic medium to an anisotropic medium. In the first part of this work, I examine regional changes in seismic response due to the production of oil and gas from one reservoir. The previous studies primarily used two post-production ocean-bottom surveys (Phase I and Phase II), and not the legacy streamer data, due to the unavailability of legacy prestack data and very different acquisition parameters. In order to incorporate the legacy data in the present study, all three poststack data sets were cross-equalized and examined using instantaneous amplitude and energy volumes. This approach appears quite effective and helps to suppress changes unrelated to production while emphasizing those large-amplitude changes that are related to production in this noisy (by current standards) suite of data. I examine the multiple data sets first by using the instantaneous amplitude and energy attributes, and then also examine specific apparent time-lapse changes through direct comparisons of seismic traces. In so doing, I identify time-delays that, when corrected for, indicate water encroachment at the base of the producing reservoir. I also identify specific sites of leakage from various unproduced reservoirs, the result of regional pressure blowdown as explained in previous studies; those earlier studies, however, were unable to identify direct evidence of fluid movement. Of particular interest is the identification of one site where oil apparently leaked from one reservoir into a “new” reservoir that did not originally contain oil, but was ideally suited as a trap for fluids leaking from the neighboring spill-point. With continued pressure drop, oil in the new reservoir increased as more oil entered into the reservoir and expanded, liberating gas from solution. Because of the limited volume available for oil and gas in that temporary trap, oil and gas also escaped from it into the surrounding formation. I also note that some of the reservoirs demonstrate time-lapse changes only in the “gas cap” and not in the oil zone, even though gas must be coming out of solution everywhere in the reservoir. This is explained by interplay between pore-fluid modulus reduction by gas saturation decrease and dry-frame modulus increase by frame stiffening. In the second part of this work, I examine various rock-physics models in an attempt to quantitatively account for frame-stiffening that results from reduced pore-fluid pressure in the producing reservoir, searching for a model that would predict the unusual AVO features observed in the time-lapse prestack and stacked data at Teal South. While several rock-physics models are successful at predicting the time-lapse response for initial production, most fail to match the observations for continued production between Phase I and Phase II. Because the reservoir was initially overpressured and unconsolidated, reservoir compaction was likely significant, and is probably accomplished largely by uniaxial strain in the vertical direction; this implies that an anisotropic model may be required. Using Walton’s model for anisotropic unconsolidated sand, I successfully model the time-lapse changes for all phases of production. This observation may be of interest for application to other unconsolidated overpressured reservoirs under production.

Time-lapse pressure tomography for characterizing CO2 plume evolution in a deep saline aquifer

A time-lapse pressure tomography inversion approach is applied to characterize the CO2 plume development in a virtual deep saline aquifer. Deep CO2 injection leads to flow properties of the mixed-phase, which vary depending on the CO2 saturation. Analogous to the crossed ray paths of a seismic tomographic experiment, pressure tomography creates streamline patterns by injecting brine prior to CO2 injection or by injecting small amounts of CO2 into the two-phase (brine and CO2) system at different depths. In a first step, the introduced pressure responses at observation locations are utilized for a computationally rapid and efficient eikonal equation based inversion to reconstruct the heterogeneity of the subsurface with diffusivity (D) tomograms. Information about the plume shape can be derived by comparing D-tomograms of the aquifer at different times. In a second step, the aquifer is subdivided into two zones of constant values of hydraulic conductivity (K) and specific storage (Ss) through a clustering approach. For the CO2 plume, mixed-phase K and Ss values are estimated by minimizing the difference between calculated and “true” pressure responses using a single-phase flow simulator to reduce the computing complexity. Finally, the estimated flow property is converted to gas saturation by a single-phase proxy, which represents an integrated value of the plume. This novel approach is tested first with a doublet well configuration, and it reveals a great potential of pressure tomography based concepts for characterizing and monitoring deep aquifers, as well as the evolution of a CO2 plume. Still, field-testing will be required for better assessing the applicability of this approach.