Femtosecond laser nanoaxotomy lab-on-a-chip for in-vivo nerve regeneration studies

Surgery 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.