This research presents a system for mechanically characterizing single cells using automated micropipette aspiration. Using vision-based control and position control, the system controls a micromanipulator, a motorized translation stage, and a custom-built pressure system to position a micropipette (4 $\mu$m opening) to approach a cell, form a seal, and aspirate the cell into the micropipette for quantifying the cell's elastic and viscoelastic parameters as well as viscosity. Image processing algorithms were developed to provide controllers with real-time visual feedback and to accurately measure cell deformation behavior on the fly. Experiments on both solid-like and liquid-like cells demonstrated that the system is capable of efficiently performing single-cell micropipette aspiration and has low operator skill requirements. Once the system was validated, it was used to study voided urine cells. In this study, the mechanical properties of bladder carcinoma cells were investigated.

This research presents a system for mechanically characterizing single cells using automated micropipette aspiration. Using vision-based control and position control, the system controls a micromanipulator, a motorized translation stage, and a custom-built pressure system to position a micropipette (4 $\mu$m opening) to approach a cell, form a seal, and aspirate the cell into the micropipette for quantifying the cell's elastic and viscoelastic parameters as well as viscosity. Image processing algorithms were developed to provide controllers with real-time visual feedback and to accurately measure cell deformation behavior on the fly. Experiments on both solid-like and liquid-like cells demonstrated that the system is capable of efficiently performing single-cell micropipette aspiration and has low operator skill requirements. Once the system was validated, it was used to study voided urine cells. In this study, the mechanical properties of bladder carcinoma cells were investigated.

Biological sample analysis is a costly and time-consuming process. It involves highly trained technicians operating large and expensive instruments in a temperature and dust controlled environment. In the world of rising healthcare cost, the drive towards a more cost-effective solution calls for a point-of-care device that performs accurate analyses of human blood samples. To achieve this goal, today's bulky laboratory instruments need to be scaled down and integrated on a single microchip of only a few square centimeters or millimeters in size. Dielectrophoresis (DEP), a phenomenon where small particles such as human blood cells are manipulated by non-uniform electric fields, stands to feature prominently in the point-of-care device. An original device that enhances DEP effect through novel geometry of the electrodes is presented. When activated with two inverting sinusoidal waveforms, the novel-shaped electrodes generate horizontal bands of increasing electric fields on the surface of the microchip. With these bands of electric fields, particles can be manipulated to form a straight horizontal line at a predictable location. Experimental results showing the collection, separation, and transportation of mammalian cells are presented. A strategy for simultaneous processing of two or more types of particles is also demonstrated. With capabilities for an accurate position control and an increased throughput by parallel processing, the novel microchip device delivers substantial improvements over the existing DEP designs. The research presented here explores the effects of novel electrode geometries in cell manipulations and contributes to the overall progress of an automated blood analysis system.

Biological sample analysis is a costly and time-consuming process. It involves highly trained technicians operating large and expensive instruments in a temperature and dust controlled environment. In the world of rising healthcare cost, the drive towards a more cost-effective solution calls for a point-of-care device that performs accurate analyses of human blood samples. To achieve this goal, today's bulky laboratory instruments need to be scaled down and integrated on a single microchip of only a few square centimeters or millimeters in size. Dielectrophoresis (DEP), a phenomenon where small particles such as human blood cells are manipulated by non-uniform electric fields, stands to feature prominently in the point-of-care device. An original device that enhances DEP effect through novel geometry of the electrodes is presented. When activated with two inverting sinusoidal waveforms, the novel-shaped electrodes generate horizontal bands of increasing electric fields on the surface of the microchip. With these bands of electric fields, particles can be manipulated to form a straight horizontal line at a predictable location. Experimental results showing the collection, separation, and transportation of mammalian cells are presented. A strategy for simultaneous processing of two or more types of particles is also demonstrated. With capabilities for an accurate position control and an increased throughput by parallel processing, the novel microchip device delivers substantial improvements over the existing DEP designs. The research presented here explores the effects of novel electrode geometries in cell manipulations and contributes to the overall progress of an automated blood analysis system.

Integrated microfluidic devices for biomedical analysis attract lots of interest in the MEMS (Micro-Electro-Mechanical-Systems) research field. However, the characteristic Reynolds number for liquids flowing in these microchannels is very small (typically less than 10). At such low Reynolds numbers, turbulent mixing does not occur and homogenization of the solutions occurs through diffusion processes alone. Hence, a satisfactory mixing performance generally requires the use of extended flow channels and takes longer to accomplish such that the practical benefits of such devices are somewhat limited. Consequently, accomplishing the goal of u¡VTAS requires the development of enhanced mixing techniques for microfluidic structures. This study first presents a microfluidic mixer utilizing alternatively switching electroosmotic flow and proposes two microchannel designs of T-form and double-T-form micromixer. Switching DC field is used to generate the electroosmotic force to drive the fluid and also used for mixing of the fluids simultaneously, such that moving parts in the microfluidic device and delicate external control system are not required for the mixing purpose. Furthermore, this study also proposed a novel pinched-switching mode in the T-form microfluidic mixer, which could be effectively increase the perturbation within the fluid to promote the mixing efficiency. In this study, computer simulation for the operation conditions is used to predict the mixing outcomes and the mixing performance is also confirmed experimentally. Result shows the mixing performance can be as larger as 95% within the mixing distance of 1 mm downstream the common boundary between the different sample fluids. The novel method proposed in this study can be used for solving the mixing problem in a simple way in the field of micro-total-analysis-systems. Furthermore, in order to demonstrate the proposed micromixer is feasible for on-line bio-reaction, this study designs a fully integrated device for demonstration of DNA/enzyme reaction within the microfluidic chip. The microchip device contains a pre-column concentrating region, a micro mixer for DNA-enzyme mixing, an adjustable temperature control system and a post-column concentration channel. The integrated microfluidic chip has been used to implement the DNA digestion and extraction. Successfully digestion of £f-DNA using EcoRI restriction enzyme in the proposed device is demonstrated utilizing large-scale gel electrophoresis scheme. Results show that the reaction speed doubled while using the microfluidic system. In addition, on-line DNA digestion and capillary electrophoresis detection is also successfully demonstrated using a standard DNA-enzyme system of $X-174 and Hae III. Finally, this reasearch also proposes a novel cell/microparticle manipulation platform by integrating an optical tweezer system and a micro flow cytometer. During operation, electrokinetically driven sheath flows are utilized to focus microparticles to flow in the center of the sample stream then pass through an optical manipulation area. An IR diode laser is focused to generate force gradient in the optical manipulation area to manipulate the microparticles in the microfluidic device. Moving the particles at a static condition is demonstrated to confirm the feasibility of the home-built optical tweezer. The trapping force of the optical tweezer is measured using a novel method of Stocks-drag equilibrium. The proposed system can continuously catch moving microparticles in the flowing stream or switch them to flow into another sample flow within the microchannel. Target particles can be separated from the sample particles with this high efficient approach. More importantly, the system demonstrates a continuously manipulation of microparticles using non-contact force gradient such that moving parts and delicate fabrication processes can be excluded. The proposed system is feasible of high-throughput catching, moving, manipulation and sorting specific microparticles/cells within a mixed sample and results in a simple solution for cell/microparticle manipulation in the field of micro-total-analysis-systems. In this thesis, low-cost soda-lime glass substrates are adopted for the microchip fabrication using a simple and reliable fabrication process. Three kinds of novel microfluidic devices including an electrokinetically-driven microfluidic mixer, a high throughput DNA/enzyme reactor and an optically cell manipulation platform are successfully demonstrated. It is the author¡¦s believes that the results of this study will give important contributions in the development of micro-total-analysis-systems in the future. With the success of this study, we have a further step approaching to the dream of lab-on-a-chip system for bio-analytical applications.

<p>This thesis will investigate previously unexplored concepts in magnetic manipulation including controlling the assembly of magnetic and nonmagnetic particles either in bulk fluid or near a substrate. Both uniform glass interfaces and substrates with magnetic microstructures are considered. The main goal of this work is to discuss new strategies for implementing magnetic assembly systems that are capable of exquisitely controlling the positions and orientations of single-component as well as multi-component particle suspensions, including both magnetic and non-magnetic particles. This work primarily focuses on controlling spherical particles; however, there are also several demonstrations of controlling anisotropically shaped particles, such as microrods and Janus colloids. </p><p> Throughout this work, both conventional magnetophoresis and inverse magnetophoresis techniques were employed, the latter relying on ferrofluid, i.e. a suspension of magnetic nanoparticles in a nonmagnetic carrier fluid, which provides a strong magnetic permeability in the surrounding fluid in order to manipulate effectively non-magnetic materials. In each system it was found that the dimensionless ratio between magnetic energy and thermal energy could be successfully used to describe the degree of control over the positions and orientations of the particles. One general conclusion drawn from this work is that the ferrofluid can be modeled with a bulk effective permeability for length scales on the order of 100 nm. This greatly reduces modeling requirements since ferrofluid is a complex collection of discrete nanoparticles, and not a homogenous fluid. It was discovered that the effective magnetic permeability was often much larger than expected, and this effect was attributed to particle aggregation which is inherent in these systems. In nearly all cases, these interactions caused the ferrofluid to behave as though the nanoparticles were clustered with an effective diameter about twice the real diameter.</p><p> The principle purpose of this thesis is to present novel systems which offer the ability to manipulate and orient multi-component spherical or anisotropic particle suspensions near surfaces or in the bulk fluid. First, a novel chip-based technique for transport and separation of magnetic microparticles is discussed. Then, the manipulation of magnetic nanoparticles, for which Brownian diffusion is a significant factor, is explored and modeled. Parallel systems of nonmagnetic particles suspended in ferrofluid are also considered in the context of forming steady state concentration gradients. Next, systems of particles interacting with planar glass interfaces are analyzed, modeled, and a novel application is developed to study the interactions between antigen-antibody pairs by using the self-repulsion of non-magnetic beads away from a ferrofluid/glass interface. This thesis also focuses on studying the ability to manipulate particles in the bulk fluid. First, simple dipole-dipole aggregation phenomenon is studied in suspensions of both nonmagnetic polystyrene particles and endothelial cells. For the sizes of particles considered in these studies, currently accepted diffusion limited aggregation models could not explain the observed behavior, and a new theory was proposed. Next, this thesis analyzed the interactions that exist in multi-component magnetic and nonmagnetic particle suspensions, which led to a variety of novel and interesting colloidal assemblies. This thesis finally discusses the manipulation of anisotropic particles, namely, the ability to control the orientation of particles including both aligning nonmagnetic rods in ferrofluid as well as achieving near-holonomic control of Janus particles with optomagnetic traps. General conclusions of the viability of these techniques are outlined and future studies are proposed in the final chapter.</p> / Dissertation

In this Thesis, we have developed a microtechnology-based method for culturing and visualizing high numbers of individual cells and cell-cell interactions over extended periods of time. The foundation of the device is a silicon-glass multiwell microplate (also referred as microchip) directly compatible with fluorescence microscopy. The initial microchip design involved thousands of square wells of sizes up to 80 µm, for screening large numbers of cell-cell interactions at the single cell level. Biocompatibility and confinement tests proved the feasibility of the idea, and further investigation showed the conservation of immune cellular processes within the wells. Although the system is very reliable for screening, limitations related to synchronization of the interaction events, and the inability to maintain conjugations for long time periods, led to the development of a novel ultrasonic manipulation multiwell microdevice. The main components of the ultrasonic device is a 100-well silicon-glass microchip and an ultrasonic transducer. The transducer is used for ultrasonic actuation on the chip with a frequency causing half-wave resonances in each of the wells (2.0-2.5 MHz for wells with sizes 300-350 µm). Therefore, cells in suspension are directed by acoustic radiation forces towards a pressure node formed in the center of each well. This method allows simultaneous aggregation of cells in all wells and sustains cells confined within a small area for long time periods (even up to several days). The biological target of investigation in this Thesis is the natural killer (NK) cells and their functional properties. NK cells belong to the lymphatic group and they are important factors for host defense and immune regulation. They are characterized by the ability to interact with virus infected cells and cancer cells upon contact, and under suitable conditions they can induce target cell death. We have utilized the ultrasonic microdevice to induce NK-target cell interactions at the single cell level. Our results confirm a heterogeneity within IL-2 activated NK cell populations, with some cells being inactive, while others are capable to kill quickly and in a consecutive manner. Furthermore, we have integrated the ultrasonic microdevice in a temperature regulation system that allows to actuate with high-voltage ultrasound, but still sustain the cell physiological temperature. Using this system we have been able to induce formation of up to 100 solid tumors (HepG2 cells) in parallel without using surface modification or hydrogels. Finally, we used the tumors as targets for investigating NK cells ability to infiltrate and kill solid tumors.  To summarize, a method is presented for investigating individual NK cell behavior against target cells and solid tumors. Although we have utilized our technique to investigate NK cells, there is no limitation of the target of investigation. In the future, the device could be used for any type of cells where interactions at the single cell level can reveal critical information, but also to form solid tumors of primary cancer cells for toxicology studies. / <p>QC 20150113</p>

No description available.

Mechanical manipulation and characterization of biological cells have wide applications in genetics, reproductive biology, and cell mechanics. This research focuses on (1) the development of enabling microrobotic systems and techniques for automated cell microinjection and in situ mechanical characterization; and (2) the demonstration of molecule efficacy testing and cell quality assessment with the new technologies. Targeting high-speed cell injection for molecule screening, a first-of-its-kind automated microrobotic cell injection system is developed for injecting foreign materials (e.g., DNA, morpholinos, and proteins) into zebrafish embryos (~1.2 millimeter) and mouse oocytes/embryos (~100 micrometers), which overcomes the problems inherent in manual operation, such as long learning curves, human fatigue, and large variations in success rates due to poor reproducibility. Novel cell holding devices are developed for immobilizing a large number of embryos into a regular pattern, greatly facilitating sample preparation and increasing the sample preparation speed. Leveraging motion control and computer vision techniques, the microrobotic system is capable of performing robust cell injection at a high speed with high survival, success, and phenotypic rates. The mouse embryo injection system is applied to molecule testing of recombinant mitochondrial proteins. The efficacy of an anti-apoptotic Bcl-xL (Delta_TM) protein is, for the first time, quantitatively evaluated for enhancing the development competence of mouse embryos. For cell quality assessment, this research develops a vision-based technique for real-time cellular force measurement and in situ mechanical characterization of individual cells during microinjection. A microfabricated elastic device and a sub-pixel computer vision tracking algorithm together resolve cellular forces at the nanonewton level. Experimental results on young and old mouse oocytes demonstrate that the in situ obtained force-deformation data can be used for mechanically distinguishing healthy mouse oocytes from those with cellular dysfunctions. This work represents the first study that quantified the mechanical difference between young and old mouse oocytes, promising a practical way for oocyte quality assessment during microinjection.

Mechanical manipulation and characterization of biological cells have wide applications in genetics, reproductive biology, and cell mechanics. This research focuses on (1) the development of enabling microrobotic systems and techniques for automated cell microinjection and in situ mechanical characterization; and (2) the demonstration of molecule efficacy testing and cell quality assessment with the new technologies. Targeting high-speed cell injection for molecule screening, a first-of-its-kind automated microrobotic cell injection system is developed for injecting foreign materials (e.g., DNA, morpholinos, and proteins) into zebrafish embryos (~1.2 millimeter) and mouse oocytes/embryos (~100 micrometers), which overcomes the problems inherent in manual operation, such as long learning curves, human fatigue, and large variations in success rates due to poor reproducibility. Novel cell holding devices are developed for immobilizing a large number of embryos into a regular pattern, greatly facilitating sample preparation and increasing the sample preparation speed. Leveraging motion control and computer vision techniques, the microrobotic system is capable of performing robust cell injection at a high speed with high survival, success, and phenotypic rates. The mouse embryo injection system is applied to molecule testing of recombinant mitochondrial proteins. The efficacy of an anti-apoptotic Bcl-xL (Delta_TM) protein is, for the first time, quantitatively evaluated for enhancing the development competence of mouse embryos. For cell quality assessment, this research develops a vision-based technique for real-time cellular force measurement and in situ mechanical characterization of individual cells during microinjection. A microfabricated elastic device and a sub-pixel computer vision tracking algorithm together resolve cellular forces at the nanonewton level. Experimental results on young and old mouse oocytes demonstrate that the in situ obtained force-deformation data can be used for mechanically distinguishing healthy mouse oocytes from those with cellular dysfunctions. This work represents the first study that quantified the mechanical difference between young and old mouse oocytes, promising a practical way for oocyte quality assessment during microinjection.