Abstract
Femtosecond laser nanosurgery has been widely accepted as an axonal injury model, enabling nerve regeneration studies in the small model organism, Caenorhabditis elegans. To overcome the time limitations of manual worm handling techniques, automation and new immobilization technologies must be adopted to improve throughput in these studies. While new microfluidic immobilization techniques have been developed that promise to reduce the time required for axotomies, there is a need for automated procedures to minimize the required amount of human intervention and accelerate the axotomy processes crucial for high-throughput. Here, we report a fully automated microfluidic platform for performing laser axotomies of fluorescently tagged neurons in living Caenorhabditis elegans. The presented automation process reduces the time required to perform axotomies within individual worms to ∼17 s/worm, at least one order of magnitude faster than manual approaches. The full automation is achieved with a unique chip design and an operation sequence that is fully computer controlled and synchronized with efficient and accurate image processing algorithms. The microfluidic device includes a T-shaped architecture and three-dimensional microfluidic interconnects to serially transport, position, and immobilize worms. The image processing algorithms can identify and precisely position axons targeted for ablation. There were no statistically significant differences observed in reconnection probabilities between axotomies carried out with the automated system and those performed manually with anesthetics. The overall success rate of automated axotomies was 67.4±3.2% of the cases (236/350) at an average processing rate of 17.0±2.4 s. This fully automated platform establishes a promising methodology for prospective genome-wide screening of nerve regeneration in C. elegans in a truly high-throughput manner.
Highlights
Acute trauma to the nervous system affects a countless number of people worldwide, though the molecular mechanisms that regulate the nerve regeneration and degeneration processes remain largely unknown
We previously demonstrated that a microfluidic immobilization technique based on a deflectable membrane could guarantee the degree of immobilization required to perform precision laser axotomies [19]
The autonomous system successfully cut axons in average 67% of the worms loaded into the microfluidic device, and the process of automated targeting and axotomy required 17 seconds for each worm
Summary
Acute trauma to the nervous system affects a countless number of people worldwide, though the molecular mechanisms that regulate the nerve regeneration and degeneration processes remain largely unknown. Microfluidic manipulation techniques for in vivo studies typically involve mechanical trapping assisted by either tapered channels [10,11,12, 22], pressurized membranes [18, 19], pressurized membranes with the addition of suction [21] or with the addition of CO2-induced paralysis [23], exposure to a cold (4 ̊C) fluid to induce temporary paralysis [9], dielectrophoresis [17], or surface acoustic wave manipulation [16] Automation of these microfluidic platforms and the related imaging and surgery processes is necessary to enable investigation of nerve regeneration in C. elegans at high speeds. The automated microfluidic platform is capable of loading a single worm from a large population, immobilizing the isolated worm, identifying the location of the worm, neuron of interest and relative location of axon, overlapping the tightly focused femtosecond laser spot on the axon of interest, and ablating it with submicron resolution, all in an automated manner
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