Browsing by Subject "Nerve regeneration"
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Item Femtosecond laser nanoaxotomy lab-on-a-chip for in-vivo nerve regeneration studies(2010-12) Guo, Xun, doctor of mechanical engineering; Ben-Yakar, AdelaSurgery of axons in C. elegans using ultrafast laser pulses, and observing their subsequent regrowth opens a new frontier in neuroscience, since such research holds a great potential for the development of novel therapies and cures to neurodegenerative diseases. In order to make the required large-scale genetic screenings in C. elegans possible and thus obtain statistically significant biological data, an automated laser axotomy system needs to be developed. Microfluidic devices hold the promise of improved throughput by integrating different functional modules into a single chip. The first step to developing a microfluidic device for laser axotomy is to devise an on-chip worm trapping method, which maintains a high degree of immobilization to sever axons without using anesthetics. In this thesis, we present a novel method that uses a thin, deflectable PDMS membrane that individually traps worms in a microfluidic device. Axons can successfully be severed with the same accuracy as those using conventional paralyzing techniques. This device also incorporates recovery chambers for housing worms after surgery and for time-lapse imaging of axonal regrowth without the repeated use of anesthetics. Towards accomplishing an automated, high-throughput laser axotomy system, we developed an improved microfluidic design based on the same mechanical immobilization technique. This second generation device allows for serially processing of a large quantity of worms rapidly using a semi-automated system. Integrated to the opto-mechanical platform, a software program utilizing image processing techniques is developed. This semi-automated program can automatically identify the location of worms, their neuronal cell bodies, focus on the axons of interest, and align the laser beam with the axon via a PID based viso-servo feedback algorithm. Statistic data demonstrate that there is no significant difference in axonal reconnection rates between surgeries performed on-chip and using anesthetics. To improve flow control, a three-dimensional novel microfluidic valve structure is designed and fabricated. This novel valve structure allows for a complete sealing of the flow channel, without degrading optical conditions for imaging and laser ablation in the trapping area. Finally, we developed a prototypical microfluidic assembly that will eventually be able to interface a well-plate to automatically deliver population of worms from individual wells to the automated chip for axotomy. This interface consists of a microfluidic multiplexer to significantly reduce the number of solenoid valves needed to individually address each well.Item Nano opto-mechanical characterization of neuron membrane mechanics under cellular growth and differentiation(2007-05) Gopal, Ashwini; Zhang, John X.J.Neurons are among the most fundamental building blocks of modern biology and medicine. Axonal development of neurons is intimately dependent on applied mechanical tension. Local 3-D cell growing microenvironment and size-dependant mechanical stimulations can have a profound impact on the reliability of regenerated nerves, but have not been well characterized. The importance of understanding cell membrane interaction with its external mechanical environment has motivated several experiments to measure cell membrane elasticity using micro-scale strain sensors, atomic force microscopy, micropipette aspiration and optical tweezers. However, the methods employed thus far are still limited by measurement range, resolution, and probe-cell interface configuration for minimal damage to cells with small size (less than 10μm), irregular shape and fragile membranes (10-1000Pa). New tools are needed to quantify the neuron membrane properties, especially the elastic modulus, to reveal the cellular responses to mechanical stimulations. Further considerations are also needed on the compatibility of probes with materials and media in which biologically relevant studies such as nerve regeneration may be performed.Recent advancements in microfabricated multi-layer grating photonics enable non-destructive sensing and microscopy of single cells with high resolution. Here we present single-layer pitch-variable diffractive nanogratings on silicon nitride probe to measure PC12 neuron model cells during growth to investigate neuronal cell mechanics and its impact on cellular differentiation and growth. We fabricated single-layer pitch-variable diffractive nanogratings on silicon nitride probe using e-beam lithography and subsequent pattern transfer techniques. We measured the mechanical membrane characteristics of PC12 cells using the force sensors with displacement range of 10 μm and force sensitivity 8 μN/μm. Young's moduli of 425±30 Pa were measured for PC 12 cells cultured on PDMS substrate coated with collagen . We have also observed stimulation of directed neurite contraction on extended probing up to 6 μm on extended probing for a time period of 30 minutes.Item Parallelized microfluidic devices for high-throughput nerve regeneration studies in Caenorhabditis elegans(2010-08) Ghorashian, Navid; Ben-Yakar, AdelaThe nexus of engineering and molecular biology has given birth to high-throughput technologies that allow biologists and medical scientists to produce previously unattainable amounts of data to better understand the molecular basis of many biological phenomena. Here, we describe the development of an enabling biotechnology, commonly known as microfluidics in the fabrication of high-throughput systems to study nerve degeneration and regeneration in the well-defined model nematode, Caenorhabditis elegans (C. elegans). Our lab previously demonstrated how femtosecond (fs) laser pulses could precisely cut nerve axons in C. elegans, and we observed axonal regeneration in vivo in single worms that were immobilized on anesthetic treated agar pads. We then developed a microfluidic device capable of immobilizing one worm at a time with a deformable membrane to perform these experiments without agar pads or anesthetics. Here, we describe the development of improved microfluidic devices that can trap and immobilize up to 24 individual worms in parallel chambers for high-throughput axotomy and subsequent imaging of nerve regeneration in a single platform. We tested different micro-channel designs and geometries to optimize specific parameters: (1) the initial trapping of a single worm in each immobilization chamber, simultaneously, (2) immobilization of single worms for imaging and fs-laser axotomy, and (3) long term storage of worms on-chip for imaging of regeneration at different time points after the initial axon cut.