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,...
We present two microfluidic approaches for immobilizing the roundworm C. elegans on-chip. The first approach creates a CO(2) micro-environment while the second one utilizes a deformable PDMS membrane to mechanically restrict the worm's movement. An on-chip 'behavior' module was used to characterize the effect of these methods on the worm's locomotion pattern. Our results indicate that both methods are appropriate for the short-term (minutes) worm immobilization. The CO(2) method offers the additional advantages of long-term immobilization (1-2 hours) and reduced photobleaching, if fluorescent imaging during immobilization is required. We envision the use of these methods in a wide variety of biological studies in C. elegans, including cell developmental and neuronal regeneration studies.
Functional fluorescence imaging methods are widely used to study cellular physiology. When applied to small organisms, these methods suffer from low-throughput due to the laborious immobilization/stimulus delivery procedure that is typically involved during imaging. Here, we describe the development of an automated microfluidic-based platform for performing automated neuronal functional (calcium) imaging in the roundworm Caenorhabditis elegans. The platform, capable of processing tens to hundreds of worms per hour, immobilizes individual worms, delivers a chemical odor to their nose and collects calcium imaging data from single neurons without any manual intervention. We used the developed platform to obtain a large number of calcium responses from worms of different ages (212 worms were imaged in total). The calcium imaging data revealed significant difference in the responses from young and old worms, indicating that neural functionality is age-dependent. We believe that such a technology will be an essential tool for obtaining repeatable and accurate functional imaging data from a large population of worms, in order to minimize stochastic biological noise and identify statistically significant trends.
We present a novel microfabrication approach for obtaining arrays of planar, polymer-based microlenses of high numerical aperture. The proposed microlenses arrays consist of deformable, elastomeric membranes that are supported by polymer-filled microchambers. Each membrane/microchamber assembly is converted into a solid microlens when the supporting UV-curable polymer is pressurized and cured. By modifying the microlens diameter (40-60 microm) and curing pressure (7.5-30 psi), we demonstrated that it is possible to fabricate microlenses with a wide range of effective focal lengths (100-400 microm) and numerical apertures (0.05-0.3). We obtained a maximum numerical aperture of 0.3 and transverse resolution of 2.8 microm for 60 microm diameter microlenses cured at 30 psi. These values were found to be in agreement with values obtained from opto-mechanical simulations. We envision the use of these high numerical microlenses arrays in optical applications where light collection efficiency is important.
Electrical stimulation has been widely used to modulate and study the in vitro and in vivo functionality of the nervous system. Here, we characterized the effect of electrical stimulation on ASH neuron in Caenorhabditis elegans and employed it to probe the neuron's age dependent properties. We utilized an automated microfluidic-based platform and characterized the ASH neuronal activity in response to an electric current applied to the worm's body. The electrically induced ASH neuronal response was observed to be dependent on the magnitude, polarity, and spatial location of the electrical stimulus as well as on the age of the worm.
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