Femtosecond laser nanosurgery in microfluidic devices and its emerging role in nerve regeneration studies

Abstract

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...