An automated microfluidic platform for calcium imaging of chemosensory neurons in Caenorhabditis elegans.

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.

CO2 and compressive immobilization of C. elegans on-chip.

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.

A high numerical aperture, polymer-based, planar microlens array.

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.

Probing the physiology of ASH neuron in Caenorhabditis elegans using electric current stimulation.

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.