A thorough understanding of nerve regeneration in Caenorhabditis elegans requires performing femtosecond laser nanoaxotomy while minimally affecting the worm. We present a microfluidic device that fulfills such criteria and can easily be automated to enable high-throughput genetic and pharmacological screenings. Using the 'nanoaxotomy' chip, we discovered that axonal regeneration occurs much faster than previously described and surprisingly the distal fragment of the severed axon regrows in the absence of anesthetics.The understanding of the biological mechanisms of nerve regeneration and degeneration after injury holds the key to developing novel therapies for human neurodegenerative diseases. These processes can be studied in model organisms by severing axons in a controlled manner, and then observing their regrowth and functional recovery. The ideal predisposition of the nematode Caenorhabditis elegans for such studies recently became accessible by the demonstration of precise nanoaxotomy using ultrafast laser pulses 1 . However, the side effects that the chemicals used to immobilize the worms for laser nanoaxotomy might have on nerve regeneration are difficult to evaluate, unless nanosurgery can be performed in vivo without anesthetics. The environment in which both surgery and monitoring are performed should have a minimal impact on the studied organism and its biological processes. To achieve this goal, we designed a microfluidic device that allows us to sever axons in C. elegans using ultrashort laser pulses with the same high precision as we demonstrated previously 1,2 while monitoring the subsequent axonal regeneration activity.Several microfluidic devices and microelectromechanical systems (MEMS) have recently been developed for C. elegans including, Petri dish-based microfluidics 3 , microfluidic traps 4,5,6 , the "CD-worm" 7 , a shadow imaging platform 8 , microfluidic maze structures 6,9 , a cantilever force MEMS sensor 10 and a platform to capture and sort worms 11 . However no demonstration of nanosurgery on-a-chip has been reported so far.Correspondence should be addressed to A.B. (ben-yakar@mail.utexas.edu The integrated microfluidic device we designed presents several unique features that are critical for the success of in vivo nerve regeneration studies: the worms are held directly against the glass cover for ideal focusing and precise nanosurgery; the trap is adjustable to the size of the worms allowing immobilization of worms at various developmental stages (L4 to adult size); and the system integrates feeding modules and thus allows long term follow-up studies of the axotomized worms as well as their sorting and screening.The high-throughput microfluidic system integrates two separate modules (Fig. 1a), a trapping module for nanosurgery and time-lapse imaging ( Fig. 1c-e) and a feeding module for recovery of the operated worms (Fig. 1b). Follow-up imaging of injured axons and their regrowth is performed using the same trapping module. Depending on the outcome of the imaging session,...

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.

Thermal conductance measurements are performed on individual polystyrene nanowires using a novel measurement technique in which the wires are suspended between two bi-material microcantilever sensors. The nanowires are fabricated via electrospinning process. Thermal conductivity of the nanowire samples is found to be between 6.6 and 14.4 W m(-1) K(-1) depending on sample, a significant increase above typical bulk conductivity values for polystyrene. The high strain rates characteristic of electrospinning are believed to lead to alignment of molecular polymer chains, and hence the increase in thermal conductivity, along the axis of the nanowire.

This paper presents an experimental study on the femtosecond (fs) laser ablation of bundles of single wall carbon nanotubes (SWCNTs) deposited on glass and the resulting nanoablation of glass beneath the bundles. The peak ablation threshold of SWCNT bundles is 50 ± 12 mJ cm −2 , which is about ten times lower than the theoretical ablation threshold of individual SWCNTs. Nanoscale ablation of the glass surface (30-50 nm wide, 20-50 nm deep and micrometres long) directly beneath the bundles is possible at a laser fluence of 920 ± 76 mJ cm −2 , which is 4-5 times lower than the fs laser ablation threshold of glass. We attribute these reduced ablation thresholds to the enhancement of fs laser pulses in the near-field of nanotube bundles. This nanoablation approach can be used for lithographical and surgical applications requiring nanoscale precision.

Fs-laser nanosurgery as a precise injury tool has opened new frontiers in achieving a thorough understanding of nerve regeneration in model organisms. Using microfluidic devices we can now perform high-throughput genetic/pharmacological screenings in Caenorhabditis elegans.The understanding of the biological mechanisms of nerve regeneration and degeneration after injury holds the key to developing new therapies for human neurodegenerative diseases. These processes can be studied in model organisms by severing axons in a controlled manner and then observing their regrowth and functional recovery. We have recently demonstrated that femtosecond (fs) laser pulses can be used as precise cutting tools for severing axons in C. elegans and that the operated axons can regenerate and recover their functionality after the operation [1]. This first demonstration of the existence of spontaneous nerve regeneration in a model organism has opened a new frontier for the nerve regeneration studies in small model organisms that can be performed using the highly precise and non-invasive cutting tool "fs-laser nanoaxotomy". There is a great interest in studying nerve regeneration in the model organism roundworm C. elegans because of its simplicity as an invertebrate organism having only 302 neurons and also because of the availability of extensive genetic tools. However, the side effects that the chemicals used to immobilize the worms for laser nanoaxotomy might have on nerve regeneration are difficult to evaluate unless nanosurgery can be performed in vivo without anesthetics. To minimize undesirable environmental effects during surgery and monitoring, we designed a microfluidic device that allows us to sever axons in C. elegans using ultrashort laser pulses with the same high precision as we previously demonstrated [1,2], while monitoring the subsequent axonal regeneration activity. Fig. 1. The nanoaxotomy lab-on-a-chip. (a) Overview of the chip with the trap system (yellow rectangle) and three recovery chambers (blue rectangle). (b) Magnified view of recovery chambers. (c) Magnified view of the trapping system. Valves 1 to 4 (yellow rectangles) respectively control inlet regulation, fine positioning of the worm (2 & 3) and gating to the recovery chambers. (d) Conceptual 3D sectional renderings of the bilayer trap channels without and with an immobilized worm. (e) Two-photon images of cross-sectional profiles of the microchannel in the trap area for increasing air pressures from 0 to 35, 70, 105, 140 and 175 kPa. (f) Cross-sectional two-photon images of a trapped worm at 105 and 140 kPa. Scale bars are 2 mm in (a) and (b), 1 mm in (c), and 50 µm in (e) and (f).Fig. 2. Nanoaxotomy on-a-chip. (a) White light picture of the trap before positioning a worm. (b) White light picture of a trapped worm (at the tip of the arrow). (c) Fluorescence image of the GFP labeled ALML axon while the body of the worm is still visible in the white light. (d) Fluorescence image of the ALML axon before axotomy with the white light turned off. (e) Fluores...

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