Information Fusion for Improved Motion Estimation

studentship award number 98318229Motion Estimation is an important research field with many commercial applications including surveillance, navigation, robotics, and image compression. As a result, the field has received a great deal of attention and there exist a wide variety of Motion Estimation techniques which are often specialised for particular problems. The relative performance of these techniques, in terms of both accuracy and of computational requirements, is often found to be data dependent, and no single technique is known to outperform all others for all applications under all conditions. Information Fusion strategies seek to combine the results of different classifiers or sensors to give results of a better quality for a given problem than can be achieved by any single technique alone. Information Fusion has been shown to be of benefit to a number of applications including remote sensing, personal identity recognition, target detection, forecasting, and medical diagnosis. This thesis proposes and demonstrates that Information Fusion strategies may also be applied to combine the results of different Motion Estimation techniques in order to give more robust, more accurate and more timely motion estimates than are provided by any of the individual techniques alone. Information Fusion strategies for combining motion estimates are investigated and developed. Their usefulness is first demonstrated by combining scalar motion estimates of the frequency of rotation of spinning biological cells. Then the strategies are used to combine the results from three popular 2D Motion Estimation techniques, chosen to be representative of the main approaches in the field. Results are presented, from both real and synthetic test image sequences, which illustrate the potential benefits of Information Fusion to Motion Estimation applications. There is often a trade-off between accuracy of Motion Estimation techniques and their computational requirements. An architecture for Information Fusion that allows faster, less accurate techniques to be effectively combined with slower, more accurate techniques is described. This thesis describes a number of novel techniques for both Information Fusion and Motion Estimation which have potential scope beyond that examined here. The investigations presented in this thesis have also been reported in a number of workshop, conference and journal papers, which are listed at the end of the document

Strategies to overcome interferences during biomass monitoring with dielectric spectroscopy

Dielectric spectroscopy is extensively used to measure the level of viable biomass during fermentations but can suffer from interference by a variety of factors including the presence of dead cells, bubbles, electric and magnetic fields, changes in the medium composition, conductivity changes and the presence of non-cellular particles. Three different approaches were used to overcome these problems. The first involved the separate measurement of the spectra of the interferent and the cells. If the spectra were significantly different then spectra containing the signals of both cells and the interferent could be deconvoluted to separately determine the relative contribution of the cells and the interferent to the spectra. This deconvolution approach was successfully used to estimate the biomass levels of yeast in the presence of spent grains of barley and hardwood in the medium. A similar approach allowed the interference of electrode polarisation on spectra of yeast and microalgae to be compensated for. An attempt to determine the concentration of non-viable cells in a mixture of dead and live cells was less successful because the signal of the non-viable cells was quite small compared to that of viable cells. A second approach involved the use of a filter to keep the interferent away from the probe surface. This was used successfully in the measurement of the yeast concentration in the presence of spent barley grains. A third approach involved the use of a second sensor in addition to the biomass sensor. This allows the signal of the biomass sensor to be compensated for the interferent. In one set of experiments microelectrodes were developed which were able to confine the electric field to a small volume near the electrode surface. Covering the electrode surface with a gel or a membrane stopped cells from entering this volume whilst allowing medium to diffuse through. This allowed the measurement of changes in the electrical properties of the medium without a contribution by the cells. Whilst this approach worked, the response time was too long for practical use. More successful was the simultaneous measurement of the biomass with an infrared optical probe and a dielectric probe. It was found that the signal of the optical probe was independent of the cell viability, whilst the dielectric probe was quite insensitive to non-viable cells. The combined use of the dielectric probe and the optical probe allowed the culture viability to be determined in a straightforward manner

REVERSE INSULATOR DIELECTROPHORESIS: UTILIZING DROPLET MICROENVIRONMENTS FOR DISCERNING MOLECULAR EXPRESSIONS ON CELL SURFACES

Lab-on-a-chip (LOC) technologies enable the development of portable analysis devices that use small sample and reagent volumes, allow for multiple unit operations, and couple with detectors to achieve high resolution and sensitivity, while having small footprints, low cost, short analysis times, and portability. Droplet microfluidics is a subset of LOCs with the unique benefit of enabling parallel analysis since each droplet can be utilized as an isolated microenvironment. This work explored adaptation of droplet microfluidics into a unique, previously unexplored application where the water/oil interface was harnessed to bend electric field lines within individual droplets for insulator dielectrophoretic (iDEP) characterizations. iDEP polarizes particles/cells within non-uniform electric fields shaped by insulating geometries. We termed this unique combination of droplet microfluidics and iDEP reverse insulator dielectrophoresis (riDEP). This riDEP approach has the potential to protect cell samples from unwanted sample-electrode interactions and decrease the number of required experiments for dielectrophoretic characterization by ~80% by harnessing the parallelization power of droplet microfluidics. Future research opportunities are discussed that could improve this reduction further to 93%. A microfluidic device was designed where aqueous-in-oil droplets were generated in a microchannel T-junction and packed into a microchamber. Reproducible droplets were achieved at the T-junction and were stable over long time periods in the microchamber using Krytox FSH 157 surfactant in the continuous oil FC-40 phase and isotonic salts and dextrose solutions as the dispersed aqueous phase. Surfactant, salts, and dextrose interact at the droplet interface influencing interfacial tension and droplet stability. Results provide foundational knowledge for engineering stable bio- and electro-compatible droplet microfluidic platforms. Electrodes were added to the microdevice to apply an electric field across the droplet packed chamber and explore riDEP responses. Operating windows for droplet stability were shown to depend on surfactant concentration in the oil phase and aqueous phase conductivity, where different voltage/frequency combinations resulted in either stable droplets or electrocoalescence. Experimental results provided a stability map for strategical applied electric field selection to avoid adverse droplet morphological changes while inducing riDEP. Within the microdevice, both polystyrene beads and red blood cells demonstrated weak dielectrophoretic responses, as evidenced by pearl-chain formation, confirming the preliminary feasibility of riDEP as a potential characterization technique. Two additional side projects included an alternative approach to isolate electrode surface reactions from the cell suspension via a hafnium oxide film over the electrodes. In addition, a commercially prevalent water-based polymer emulsion was found to adequately duplicate microchannel and microchamber features such that it could be used for microdevice replication

Optical computed tomography for spatially isotropic four-dimensional imaging of live single cells

abstract: Quantitative three-dimensional (3D) computed tomography (CT) imaging of living single cells enables orientation-independent morphometric analysis of the intricacies of cellular physiology. Since its invention, x-ray CT has become indispensable in the clinic for diagnostic and prognostic purposes due to its quantitative absorption-based imaging in true 3D that allows objects of interest to be viewed and measured from any orientation. However, x-ray CT has not been useful at the level of single cells because there is insufficient contrast to form an image. Recently, optical CT has been developed successfully for fixed cells, but this technology called Cell-CT is incompatible with live-cell imaging due to the use of stains, such as hematoxylin, that are not compatible with cell viability. We present a novel development of optical CT for quantitative, multispectral functional 4D (three spatial + one spectral dimension) imaging of living single cells. The method applied to immune system cells offers truly isotropic 3D spatial resolution and enables time-resolved imaging studies of cells suspended in aqueous medium. Using live-cell optical CT, we found a heterogeneous response to mitochondrial fission inhibition in mouse macrophages and differential basal remodeling of small (0.1 to 1 fl) and large (1 to 20 fl) nuclear and mitochondrial structures on a 20- to 30-s time scale in human myelogenous leukemia cells. Because of its robust 3D measurement capabilities, live-cell optical CT represents a powerful new tool in the biomedical research field

Microfluidic and Electrokinetic Manipulation of Single Cells

Traditional cell assays report on the average results of a cell population. However, a wide range of new tools are being developed for a fundamental understanding of single cell's functionality. Nonetheless, the current tools are either limited in their throughput or the accuracy of the analysis. One such technology is electrorotation. Although it is known to be unique in its capability for single-cell characterization, it is commonly a slow technique with a processing time of about 30 minutes per cell. For this reason, this thesis focuses on the development of a 3D electrode based electrorotation setup for fast and automatic extraction of a single cell's spectrum. For this purpose, new fabrication processes for 3D electrodes were developed to achieve high-resolution patterning of 3D metal electrodes. The first process we developed was a subtractive one based on passivated silicon structures and the second process was an additive one based on SU-8 photolithography. The additive nature of the second process enables high patterning resolution of electrodes and connection layers, while providing high conductivity thanks to the use of standard metal films. The electrodes have been characterized by different electrical measurements to ensure a proper connection and side-wall exposure. Furthermore, we characterized and compared the sheet resistance of planar and vertical layers. A further microfabrication process was developed for integrating the electrodes into microfluidic channels. The process was designed to enable the use of high numerical aperture lenses; for that purpose, a PDMS-mediated bonding process was engineered to seal the channels with a thin glass coverslip. Moreover, the development of a process to realize microfluidic access holes on the back of the wafer reduces the footprint of the chips and facilitates access for the microscope optics. Finally, a pressure-driven system was used together with the chips to achieve high control of liquid injections and to enable fast and precise flow stop. The combination of such a system, together with the dielectrophoretic forces that can be applied by the 3D electrodes, allows accurate positioning of single cells inside the 3D electrode quadrupole. The particles can then be analyzed by electrorotation. For this purpose, a custom Labview interface was built to coordinate the full setup and to acquire a full electrorotation spectrum in less than 3 minutes

Effects of Dielectrophoresis on Growth, Viability and Immuno-reactivity of Listeria monocytogenes

Dielectrophoresis (DEP) has been regarded as a useful tool for manipulating biological cells prior to the detection of cells. Since DEP uses high AC electrical fields, it is important to examine whether these electrical fields in any way damage cells or affect their characteristics in subsequent analytical procedures. In this study, we investigated the effects of DEP manipulation on the characteristics of Listeria monocytogenes cells, including the immuno-reactivity to several Listeria-specific antibodies, the cell growth profile in liquid medium, and the cell viability on selective agar plates. It was found that a 1-h DEP treatment increased the cell immuno-reactivity to the commercial Listeria species-specific polyclonal antibodies (from KPL) by ~31.8% and to the C11E9 monoclonal antibodies by ~82.9%, whereas no significant changes were observed with either anti-InlB or anti-ActA antibodies. A 1-h DEP treatment did not cause any change in the growth profile of Listeria in the low conductive growth medium (LCGM); however, prolonged treatments (4 h or greater) caused significant delays in cell growth. The results of plating methods showed that a 4-h DEP treatment (5 MHz, 20 Vpp) reduced the viable cell numbers by 56.8–89.7 %. These results indicated that DEP manipulation may or may not affect the final detection signal in immuno-based detection depending on the type of antigen-antibody reaction involved. However, prolonged DEP treatment for manipulating bacterial cells could produce negative effects on the cell detection by growth-based methods. Careful selection of DEP operation conditions could avoid or minimize negative effects on subsequent cell detection performance

Dielectrophoretic investigations of internal cell properties

Dielectrophoresis (DEP) is a term which describes the motion of polarisable particles induced by a non-uniform electric field. It has been the subject of research into a variety of fields including nanoassembly, particle filtration and biomedicine. The application of DEP to the latter has gained significant interest in recent years, driven by the development of microfluidic “Lab-on-a-chip” devices designed to perform sophisticated biochemical processes. It provides the ability to characterise and selectively manipulate cells based on their distinct dielectric properties in a manner which is non-invasive and label free, by using electrodes which can be readily integrated with microfluidic channels. Under appropriate conditions a biological cell will experience a DEP force directing it either towards or away from concentrations in the electric field. At a so-called “crossover frequency” the cell is effectively invisible to the field resulting in no DEP force, a response typically observed in the 1 kHz to 1 MHz range. Its value is a function of cell membrane dielectric properties and has been the subject of research directed at devices capable of using it to both characterise and sort cells. The aim of this work was to investigate the behaviour of a higher frequency crossover referred to as fxo2, predicted to occur in the 1 MHz to 1 GHz range. At these frequencies the electric field is expected to penetrate the cell membrane and behave as a function of intracellular dielectric properties. Standard lithography techniques have been used to fabricate electrodes carefully designed to operate at these frequencies. The existence of fxo2 was then confirmed in murine myeloma cells, in good agreement with dielectric models derived from impedance spectroscopy. A temperature dependent decrease in its value was observed with respect to the time that cells were suspended in a DEP solution. This decrease is consistent with previous studies which indicated an efflux of intracellular ions under similar conditions. An analytical derivation of fxo2 demonstrates its direct proportionality to intracellular conductivity. Direct control of the crossover was achieved by using osmotic stress to dilute the intracellular compartment and thereby alter its conductivity. By using a fluorophore which selectively binds to potassium, a strong relationship has been demonstrated between the value of fxo2 and the concentration of intracellular potassium. Measurements of fxo2 for an unfed culture demonstrated a correlation with viability and subtle shifts in its distribution were caused by the early stages of chemically induced apoptosis

Electrical cell manipulation in microfluidic systems

This dissertation reports on the development of devices and concepts for electrical and microfluidic cell manipulation. In the present context, the term cell manipulation stands for both cell handling and cell modification. The combination of microfluidic channels with micropatterned electrodes allows for the definition of highly localised chemical and electrical environments with spatial resolution comparable to the size of a cell. The devices fabricated in the frame of this thesis employ dielectrophoretic particle handling schemes such as deflection and trapping in pressure-controlled laminar flows to bring cells to – or immobilise them at – locations where cell altering electric fields or chemicals are present. The two concepts of dielectrophoretic cell dipping and cell immersion are introduced and experimentally shown for erythrocytes dipped into Rhodamine in flow, and for individually immobilised Jurkat cells immersed by Trypan Blue. Also, in-situ membrane breakdown in high intensity AC electric fields is optically assessed by efflux of haemoglobin (haemolysis) and by influx of nucleic stains or fluorescence-enhancing ions. The most advanced experiments are on-chip medium exchange followed immediately by electropermeablisation or electrodeformation. The majority of assays presented in this thesis are carried out in microfabricated glass-polymer-glass chips featuring top-bottom electrodes. The devices are fluidically controlled by external gas pressure bridging circuits. Experimental evidence of the unmatched precision of pressure bridging is given in the case of micrometric xy positioning of cells at the intersection of two perpendicular microfluidic channels. Further shown in this document are two methods of optical in-situ temperature measurements, important for bioinstrument characterisation. The two concepts of thermoquenching of a fluorescent dye and the original thermoprecipitation of "smart polymers" are used. The last part of this work deals with the innovative, conceptual engineering tool Liquid Electrode. The general concept and its advantages over solid-state electrodes are given, followed by numerical particle tracking in the case of the novel lateral nDEP particle deflection. The chapter on liquid electrodes concludes with preliminary experimental results of buffer swapping of cells in flow and of AC electropermeabilisation of erythrocytes at frequencies far below the cut-off frequency of corresponding solid-state microelectrodes