Cancer is among the leading causes of death worldwide. In 2012, there were 14 million new cases and 8.2 million cancer-related deaths worldwide. The number of new cancer cases is expected rise to 22 million within the next two decades. Most chronic cancers cannot be cured. However, if the precise cancer cell type is diagnosed at an earlier, less aggressive stage then the chance of curing the disease increases with accurate drug delivery. This work is a humble contribution to the advancement of cancer research. This work delves into biological cell phenotyping under a dielectrophoresis setup using computer vision. Dielectrophoresis is a well-known phenomenon in which dielectric particles are subjected to a non-homogeneous electric field. This work is an analytical part of a larger proposed system replete with hardware, software and microfluidics integration to achieve cancer cell characterization, separation and enrichment using contactless dielectrophoresis. To analyze the cell morphology, various detection and tracking algorithms have been implemented and tested on a diverse dataset comprising cell-separation video sequences. Other related applications like cell-counting and cell-proximity detection have also been implemented. Performances were evaluated against ground truth using metrics like precision, recall and RMS cell-count error. A detection approach using difference of Gaussian and super-pixel algorithm gave the highest average F-measure of 0.745. A nearest neighbor tracker and Kalman tracking method gave the best overall tracking performance with an average F-measure of 0.95. This combination of detection and tracking methods proved to be best suited for this dataset. A graphical user interface to automate the experimentation process of the proposed system was also designed. / Master of Science

Trapping ability on a biochip device is useful for systematic cell addressing and real-time observation of single cells analysis, however, precise control over the cell movements remains challenging. This thesis addresses the problem of controlling movement of single cells on a biochip platform by a technique called the Dielectrophoretic (DEP) force. Existing researches showed that the DEP force offers precise control of cell movements through various microelectrode designs which generate strong polarization effects i.e., DEP forces, but with the expense of damaging cell’s structure. The thesis contribute three new microelectrode designs for trapping single cells: the dipole, the quadrupole and the adaptive octupole, structured on a metal-insulator-metal (multilayer) biochip platform called the Sandwiched Insulator with Back Contact (SIBC) biochip. Cores of the study lie on the microelectrode designs that are capable of generating strong DEP holding forces, the back contact to enhance trapping of single cells and the fabrication process of creating a metal-insulator-metal structure. This thesis also presents details on the experimental setups of the trapping experiments and the numerical analysis of the microelectrode designs. The SIBC biochip comprises of the back contact on the first metal layer, the microcavity (cell trap) on the insulator layer and the three microelectrodes on the second metal layer. Together, the three microelectrodes and the back contact generate DEP forces that attract particles/single cells toward microcavities and maintain their positioning in the traps. Prior to the fabrication, profiles of the DEP force generated by the microelectrodes are studied using COMSOL3.5a software. Simulation results suggest that the DEP trapping region can be created surrounding the microcavity if the microelectrode and the back contact are connected with AC signals that have different phases. The strongest DEP force can be obtained by setting the back contact and the microelectrodes with AC signals that have 180 degree phase difference. Evaluations on the trapping functionality for the three microelectrodes were conducted using polystyrene microbeads and Ishikawa cancer cells line suspended in various medium. Trapping capability of the three microelectrodes was demonstrated through experiments with 22 percent of the Ishikawa cancer cells and 17 percent of the polystyrene microbeads were successfully trapped. With these promising results, the new microelectrode designs together with the SIBC biochip structure have huge potentials for biomedical applications particularly in the field of diagnosis and identification of diseases.

Cloning by nuclear transfer using mammalian somatic cells has enormous potential application. However, cloning mammalian species through somatic cell nuclear transfer has been simply inefficient in all species in which live clones have been produced, such as ‘Dolly’ the sheep, and ‘Samrupa’ the buffalo. Most of the experiments resulted failure, and the success rate ranges from 0.1% to 3%. Developmental defects have been attributed to incomplete reprogramming of the somatic nuclei by the cloning process. Researchers have tried strategies to improve the efficiency of nuclear transfer. However, significant breakthroughs are yet to happen. The enucleation procedure consisting of extracting reprogrammable genetic material during nuclear transfer has been linked to inefficiencies due to manual error, lack of repeatability and decreased high throughput. Conventional manual enucleation process requires a series of complicated cell rotation in three-dimensional (3D) spaces using a blunt or sharp tipped pipette, and can puncture the cell during genetic material extraction. Current methods frequently damage the cell via physical or chemical contact, and thus have low throughput. Therefore, there is a need for simple, readily automated, non-contact methods for controlled cell rotation. Precise rotation of the suspended cells is one of the many fundamental manipulations in a wide range of biotechnological applications, such as cell injection and enucleation. Noticeably scarce from the existing rotation techniques is 3D rotation of cells on one single chip. To bridge this gap, this research presents a means of controlled cell rotation for bovine oocytes around both the in-plane (yaw) and out-of-plane (pitch) axes using a simple, low cost biochip fabricated using a mixture of conventional lithography and low-cost micro-milling. It uses a phase varying dielectrophoresis (DEP)-based electrorotation (EROT) biochip platform, which has an open-top sub-millimetre square chamber enclosed by four sidewall electrodes and two bottom electrodes to induce torque to rotate the cells about two axes, thus 3D cell rotation for the first time. Before fabrication, phase varying DEP-based rotational electric field simulations were carried out in the designed rotation chamber. For this analysis, initial rotational fields are characterised for both in-plane and out-of-plane axes using multi-physics finite element software. Electrode shape and chamber design were optimised using realistic parameters for the medium and electrode material properties. Results showed remarkable promise to rotate dielectric particles in rotational field strengths of the order of 104 V/m. From simulations, a basic biochip design was optimised. Within the fabricated biochip, controlled rotations around the in-plane and out-of-plane axes were demonstrated, and the electric field activation frequency range and electrokinetic properties of the bovine oocytes were characterised. Rotation was measured via video image processing with data included on electronic annex. Results show controllable rotation in steps of 5 degrees around both axes with the same chip. In experiments, the maximum rotation rate reached 150°/s in yaw axis and 45-50°/s during pitch axis, while a smooth, stable and controllable rotation rate was found below 30-40°/s. Optimum rotation rates are found for inputs of 10 Vp-p at 500-800 kHz AC frequency for yaw-axis rotation, and 10-20 Vp-p and 10-100 kHz for pitch-axis rotation. In addition, zona intact and zona free oocytes are shown to have electrical equivalence and found no noticeable difference, generalising the bio-chips capability and results. Further, experimental results were used to validate the numerical solid shell model used in design and it was found that the bovine oocytes are highly polarizable than the surrounding medium. Finally, the dielectric properties of the oocytes were fully characterised enabling further design optimization in future, if desired. The biochip was successfully designed, optimised and experimentally validated, and successful rotation of bovine oocytes in 3D spaces was demonstrated. These results create a platform tool for biologists to utilise enucleation with high throughput efficiency and ease. In summary, this simple, transparent, low-cost, open-top, and biocompatible biochip platform, allows further function modules to be integrated and is the foundation for more powerful cell manipulation systems. In brief key novel aspects of the research were: • Rotation of suspended spherical oocytes in multiple axes (3D rotation) was obtained by AC induced electric fields. • An open top biochip was successfully fabricated to enable further processing of the rotated cell in 3D spaces. • Bovine oocyte dielectric spectra were analysed in both in-plane and out-of-plane axes for the first time. • Bovine oocytes were determined to behave as solid spherical spheres, rather than single spherical shells.

This thesis describes for the first time, how the membrane capacitance of pluripotent human embryonic stem cells (H1, H9, RCM1) increases with their differentiation (H1-MSC, H9-MSC, RCM1-trophoblast) based on the literature review. The method used to determine membrane capacitance was dielectrophoresis (DEP), which is an electrokinetic technique capable of characterising and sorting cells without the need for antibody-based cell surface markers, magnetic beads, or other chemical tags. This finding has potential biomedical importance because human embryonic stem cell (hESCs) isolated from early blastocyst-stage embryos and differentiated progeny have been identified to be of possible use in drug screening and regenerative cell based therapeutic treatment. Current cell sorting methods require membrane surface markers that limit their applicability in stem cell therapeutics, a limitation that is either removed or reduced if DEP-based sorting was used. The work described in this thesis consists of the design, fabrication and testing of DEP based microfluidic devices for characterization and separation of human embryonic stem cells. The cells studied were human undifferentiated hESC lines (H1, H9, RCM1, RH1, and T8) and their differentiated progeny (H1-MSC, H9-MSC, RCM1-trophoblast, hES-MP). The cell membrane capacitance (Cm) of the cells was determined by measuring a parameter known as the DEP cross-over frequency (fxo), where the electrical polarisability of a cell equals that of its suspending electrolyte and so experiences no DEP force. The studies of hESC lines cultured from different sources indicate, on the basis of their similar Cm values, that they have similar membrane morphologies. The change in calculated Cm value upon differentiation of these hESCs indicates that changes occur in their membrane morphology, texturing and possibly of their membrane thickness. Subsequent enrichment of these hESCs from human dermal fibroblasts (hDFs) has been achieved based on fxo measurements. The results presented in this thesis confirm the existence, previously indicated in the literature, of distinctive parameters for undifferentiated and differentiating cells on which future application of DEP in hESC manufacturing can be based.

"The importance of two-phase heat transfer for thermal management of aerospace avionic systems has become increasingly important as these systems have become miniaturized. Embedded active cooling systems are used to remove heat from processors and other electronic components and transferring this heat to radiators or other heat exchangers. As the characteristic dimension of flow channels for two-phase flow becomes comparable to bubble size, the mini-channels (< 3 mm) used to direct the cooling fluid can complicate nucleate boiling heat transfer. Bubbles can encounter other heated walls, rapidly expanding and greatly reducing heat transfer as well as causing pressure oscillations and flow instabilities. The use of eletrohydrodynamic (EHD) effects, through the introduction of non-uniform electric fields, can help mitigate this problem by altering the behavior of nucleating bubbles. A combined experimental and computational study was undertaken using HFE-7100, an engineered fluid used in heat transfer applications, to investigate the potential for enhancement of nucleate boiling using EHD effects induced by applying a non-uniform electric field. In the experimental study, a minichannel was constructed consisting of an upper and lower copper electrode and glass side walls to allow visualization. The channel height and width were 3mm and 4.76 mm respectively, representative of the minichannel regime. The upper electrode was grounded while the lower electrode was heated and biased to high voltage. Optical imaging combined with post-processing and statistical analysis was used to quantify the effect of EHD on the bubble behavior. Bubbles were found to form preferentially on nucleation sites resulting from imperfections in the heated copper surface over artificially created nucleation sites. When a high voltage is applied across the electrodes, the electric field enhancement along the rim of the nucleation site is believed to influence the force balance on the forming bubble and thereby influence the bubble departure size and frequency. EHD forces also act on the bubble surface as a result of the variation in permittivity between the liquid and vapor phases, altering its shape as has been previously reported in the literature. Test results are presented that demonstrate that the application of EHD increases the nucleation site density on the heated surface and increase the bubble departure frequency from individual sites. In addition, test results are presented to show that EHD forces alter the shape of bubbles during growth and the vertical position of the detached bubbles as they are carried along in the cross flow. To better understand the underlying phenomena affecting the bubble shape and departure frequency, a numerical simulation of the bubble growth and departure was performed using COMSOL multiphysics software customized to incorporate a user-defined body force based on the Maxwell Stress Tensor. Tracking of the bubble surface, including coalescence and breakup was incorporated using the phase field variable method in which the Navier-Stokes and heat transfer equations are solved for each phase of the fluid. Results from the simulations confirmed the sensitivity of the bubble elongation and neck formation to the nucleation site geometry, specifically the angle along the rim where field enhancement occurs. The enhanced constriction of the bubble neck resulted in early detachment of bubbles when compared to simulations in which EHD was not applied. This finding provides some insight into the higher bubble departure frequency and nucleation site density observed in the experiment. The results from the combined experimental and numerical study suggest that EHD enhancement may provide a mechanism for extending the use of nucleate heat transfer to minichannels, thereby enabling additional options for cooling in compact, embedded systems. "

Huang Ji Ping. / "1st June, 2003." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (p. 125-134). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

La cytométrie en flux en association avec la coloration et le marquage d'anticorps présente l'un des outils les plus précieux en biotechnologie actuelle fournissant des informations sur l'hétérogénéité des populations cellulaires, la taille et le volume des cellules, ainsi que l'expression de certaines molécules de surface et intracellulaires. L'augmentation du coût et la difficulté fondamentale de ces méthodes, cependant, sont attribués à l'exigence des molécules de marquage de surface. Diélectrophorèse (DEP) a été identifiée comme alternative sans marquage prometteuse. Cette thèse porte sur l'amélioration des technologies basée sur les DEP actuelles, et le développement d'une nouvelle méthode pour aborder les questions de cytometrie diélectrophorétique (DEP) permettant la mesure probabiliste des signatures diélectriques (DE) de cellules au niveau d’une population, ainsi que de permettre l'identification de biomarqueurs fiables pour les changements cellulaires.Tout d'abord, les améliorations de la cytométrie DEP sur la translation de cellules latérales induites par DEP sont explorées par fabrication. Un système de tri cellulaire benchmark microfluidique est présenté, et l'effet des désalignements des microcanaux sur les topologies des électrodes des cellules DEP vivantes est discuté. Un modèle de S. cerevisiae est présenté et validé expérimentalement dans des dispositifs microfluidiques fabriqués. Un nouveau procédé de fabrication permettant le prototypage rapide de dispositifs microfluidiques avec des électrodes intégrées bien alignées est présenté. Des dispositifs identiques ont été fabriqués avec des procédés standards de lithographie douce PDMS. Selon l'étude benchmark, la procédure standard PDMS est tombée bien en dessous de la gamme nécessaire pour le tri des cellules par DEP. Le temps de fabrication et les coûts de la méthode proposée se sont révélés être à peu près les mêmes.Deuxièmement, une nouvelle méthode appelée cytométrie DEP distribuée (2DEP cytométrie) a été développée. Elle utilise une translation verticale de cellules induite par effet de DEP en liaison avec la vélocimétrie par image de particules (PIV) afin de mesurer la répartition probabiliste de forces DEP sur une population cellulaire entière. La méthode a été intégrée dans un dispositif microfluidique avec des électrodes intégrées. Les cellules passant à travers le micro-canal sont sollicitées par des forces de sédimentation, tandis que les forces DEP soit s’opposent à la sédimentation, prennent en charge la sédimentation, ou aucun des deux, en fonction des signatures DE des cellules. Les hauteurs à laquelle les cellules se stabilisent correspondent à leur signature DE et sont mesurées indirectement en utilisant PIV.Les données expérimentales quantifient la signature DE d'une population de S. cerevisiae et la lignée cellulaire human immortalise leucemie myeloide K562. Tout d'abord, l'effet de la surexpression de certaines protéines membranaires a été étudié dans des cellules S. cerevisiae. La répartition mesurée des forces DEP a été comparée à la population de cellules exprimant une protéine cytoplasmique au même taux. Deuxièmement, 2DEP cytométrie a été appliquée à la lignée cellulaire K562. Les effets de la réponse à un stress provoqué par divers inducteurs sur la signature DE de la population cellulaire ont été analysées.Enfin, l'analyse statistique des données définies estimation par noyau ajustées pour surmonter la nature finie des données mesurées. En combinaison avec des spectres en distance de Wasserstein, notés signatures Wasserstein, ont été quantifiés et liée à certains changements cellulaires. Ces signatures peuvent être utilisées comme marqueurs biologiques fiables pour certains changements cellulaires.En conclusion, 2DEP cytométrie a montré être suffisamment sensible pour identifier certains changements d’états cellulaires. Le nouveau dispositif 2DEP cytométrie est donc une alternative prometteuse à la cytométrie en flux classique / Flow cytometry in combination with staining and antibody labelling presents one of the most valuable tools in current biotechnology providing information about cell population heterogeneity, cell size and volume, as well as expression of certain surface and intracellular molecules. The increased cost and the fundamental difficulty of these methods, however, are attributed to the requirement of the surface marker molecules. Attractive alternatives to flow cytometry are label-free methods, such as micro-filtration, dielectric spectroscopy, and electro-kinetic methods. Out of these methods, dielectrophoresis (DEP) was selected as the most promising approach. This thesis focuses on improvements of current DEP-based technologies, and development and establishment of a new method to address the issues of dielectrophoretic (DEP) cytometry enabling label-free non-invasive probabilistic measurement of cell dielectric (DE) signatures on population level, as well as enabling identification of reliable biomarkers for cell changes.First, improvements of DEP cytometry based on DEP-induced lateral cell translation through fabrication are explored. A benchmark microfluidic live cell sorting system is presented, and the effect of microchannel misalignment above electrode topologies on live cell DEP is discussed in detail. Simplified model of budding S. cerevisiae cell is presented and validated experimentally in fabricated microfluidic devices. A novel fabrication process enabling rapid prototyping of microfluidic devices with well-aligned integrated electrodes is presented and the process flow is described. Identical devices were produced with standard PDMS soft lithography processes. The presented fabrication process significantly improved the alignment of the microstructures. According to the benchmark study, the standard PDMS procedure fell well outside the range required for reasonable cell sorting efficiency. The fabrication time and costs of the proposed methodology were found to be roughly the same.Second, a method called distributed dielectrophoretic cytometry (2DEP cytometry) was developed. It uses a DEP-induced vertical translation of live cells in conjunction with PIV in order to measure probabilistic distribution of live cell DE signatures on an entire cell population. The method was integrated in a microfluidic device with integrated electrodes. Cells passing through the microchannel are acted on by sedimentation forces, while DEP forces either oppose sedimentation, support sedimentation, or neither, depending on the DE signatures of the cells. The heights at which cells stabilize correspond to their DE signature and are measured indirectly using particle image velocimetry (PIV).Experimental data quantify the DE signature of a S. cerevisiae population and Human immortalised myelogenous leukaemia cell line K562. First, the effect of over-expression of certain membrane protein was studied in S. cerevisiae cells. Measured distribution of DEP forces was compared to cell population expressing a cytoplasmic protein at the same rate. Second, 2DEP cytometry was applied to K562 cell line. Effects of stress response triggered by various inducers on the DE signature of the cell population were analysed.Finally, statistical data analysis defined adjusted kernel density estimation to overcome the finite nature of the measured data. In combination with Wasserstein pseudometrics from sampled data, the Wasserstein distance spectra, denoted as Wasserstein signatures, were quantified and linked to certain cell changes. These signatures may be used as reliable biomarkers for cell changes.In conclusion, 2DEP cytometry showed it is sensitive enough to identify certain changes in cell states. The novel 2DEP cytometry device is therefore a promising alternative to conventional flow cytometry

During the last decade, large and costly instruments are being replaced by system based on microfluidic devices. Microfluidic devices hold the promise of combining a small analytical laboratory onto a chip-sized substrate to identify, immobilize, separate, and purify cells, bio-molecules, toxins, and other chemical and biological materials. Compared to conventional instruments, microfluidic devices would perform these tasks faster with higher sensitivity and efficiency, and greater affordability. Dielectrophoresis is one of the enabling technologies for these devices. It exploits the differences in particle dielectric properties to allow manipulation and characterization of particles suspended in a fluidic medium. Particles can be trapped or moved between regions of high or low electric fields due to the polarization effects in non-uniform electric fields. By varying the applied electric field frequency, the magnitude and direction of the dielectrophoretic force on the particle can be controlled. Dielectrophoresis has been successfully demonstrated in the separation, transportation, trapping, and sorting of various biological particles. / Singapore-MIT Alliance (SMA)

Thesis (M.S.)--West Virginia University, 2008. / Title from document title page. Document formatted into pages; contains ix, 115 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 103-105).

In this thesis, three specific research contributions to the use of non-uniform electric field driven colloidal assembly are described. The first relates to experimental work using dielectrophoretic and electrohydrodynamic forces (electroosmosis) to shape three-dimensional colloidal structures. Formation and stabilization of close-packed three-dimensional structures from colloidal silica was demonstrated, using gelation of pluronic F-127 to preserve medium structure against suspension evaporation. Stabilization of ordered structures was shown to be a significant challenge, with many of the conventional techniques for immobilizing colloidal crystals being ineffective. Secondly, the significance of electrohydrodynamic flows resulting from electric and particle concentration (entropic) gradients during the assembly process was demonstrated using numerical simulations based on a thermodynamic framework. These simulations, as well as experimental validation of assembly and the presence of fluid flows, showed that assuming equilibrium behavior (stationary fluid flow), a common assumption for most modelling work to date in these systems, is inappropriate at all but the most dilute concentration cases. Finally, the relevance of multiparticle effects on electric-field induced phase transitions of dielectric colloids was demonstrated. The effect of multiparticle/multiscattering effects on the suspension permittivity were accounted for using semi-empirical continuum permittivity formulations which have been previously shown to describe a wide variety of solid packing structures, including face-centered cubic and other colloidal crystal structures. It was shown that multiparticle effects have a significant impact on both the coexistence (slow phase separation) and spinodal (fast phase separation) behavior of dielectric suspensions, which has not been demonstrated to date using a continuum framework. / Thesis (Ph.D, Chemical Engineering) -- Queen's University, 2012-01-30 14:17:23.747