title: A fully automated microfluidic femtosecond laser axotomy platform for nerve regeneration studies in C. elegans.
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.
authors.affiliation: Electrical and Computer Engineering, University of Texas, Austin, Texas, 78712, United States of America.
authors.forename: Sertan Kutal
authors.initials: SK
authors.lastname: Gokce
authors.affiliation: Mechanical Engineering, University of Texas, Austin, Texas, 78705, United States of America.
authors.forename: Samuel X
authors.initials: SX
authors.lastname: Guo
authors.affiliation: Biomedical Engineering, University of Texas, Austin, Texas, 78705, United States of America.
authors.forename: Navid
authors.initials: N
authors.lastname: Ghorashian
authors.affiliation: Mechanical Engineering, University of Texas, Austin, Texas, 78705, United States of America.
authors.forename: W Neil
authors.initials: WN
authors.lastname: Everett
authors.affiliation: Mechanical Engineering, University of Texas, Austin, Texas, 78705, United States of America.
authors.forename: Travis
authors.initials: T
authors.lastname: Jarrell
authors.affiliation: Mechanical Engineering, University of Texas, Austin, Texas, 78705, United States of America.
authors.forename: Aubri
authors.initials: A
authors.lastname: Kottek
authors.affiliation: Electrical and Computer Engineering, University of Texas, Austin, Texas, 78712, United States of America.
authors.forename: Alan C
authors.initials: AC
authors.lastname: Bovik
authors.affiliation: Electrical and Computer Engineering, University of Texas, Austin, Texas, 78712, United States of America; Mechanical Engineering, University of Texas, Austin, Texas, 78705, United States of America; Biomedical Engineering, University of Texas, Austin, Texas, 78705, United States of America.
authors.forename: Adela
authors.initials: A
authors.lastname: Ben-Yakar
mesh_terms: D000818:Animals; D001331:Automation; D001369:Axons / Q000502:physiology; D019771:Axotomy / Q000295:instrumentation* / Q000379:methods*; D017173:Caenorhabditis elegans / Q000502:physiology*; D004867:Equipment Design; D007091:Image Processing, Computer-Assisted / Q000379:methods; D007834:Lasers; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation; D009416:Nerve Regeneration / Q000502:physiology*; D009474:Neurons / Q000502:physiology; D015203:Reproducibility of Results; D013997:Time Factors