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.

The Genetics of Axon Guidance and Axon Regeneration in Caenorhabditis elegans

“…Significant effort has been put into developing microfluidic methods for worm handling, facilitating larger scale approaches (Guo et al 2008). A fully automated platform for laser axotomy has been reported (Gokce et al 2014), and it will be interesting to see the results from these approaches.…”

Axon regeneration in C. elegans: Worming our way to mechanisms of axon regeneration

“…Thus, with this mutant, very large numbers of axons can be injured (at the expense of temporal and spatial precision), facilitating genetic and genomic screening (Hammarlund et al, 2009; Rohde et al, 2009; Nix et al, 2014). An alternative approach is to automate laser surgery, potentially increasing the number of axons that can be injured and analyzed (Guo et al, 2008; Gokce et al, 2014). The availability of diverse axon injury models in C. elegans enables researchers to select the optimal approach for a given experiment.…”

An automated microfluidic system for screening Caenorhabditis elegans behaviors using electrotaxis

“…8,9,[12][13][14][15][16][17] Moreover, microfluidic devices can be automated to handle single or a population of C. elegans in a high-throughput fashion, which enables large-scale assays such as drug and genetic screens. [15][16][17][18][19][20][21][22] In previous work from our group, we designed a simple microfluidic device to examine the swimming behaviors of C. elegans under electric fields, termed electrotaxis, and demonstrated that changes in movement are reliable indicators of neurodegeneration and harmful effects of toxic chemicals on C. elegans health. [23][24][25] However, the inherent manual operation of the devices makes the screening process labor intensive and time consuming, which limits largely the practical application of the method.…”