Laterally Orienting C. elegans Using Geometry at Microscale for High-Throughput Visual Screens in Neurodegeneration and Neuronal Development Studies

Abstract

C. elegans is an excellent model system for studying neuroscience using genetics because of its relatively simple nervous system, sequenced genome, and the availability of a large number of transgenic and mutant strains. Recently, microfluidic devices have been used for high-throughput genetic screens, replacing traditional methods of manually handling C. elegans. However, the orientation of nematodes within microfluidic devices is random and often not conducive to inspection, hindering visual analysis and overall throughput. In addition, while previous studies have utilized methods to bias head and tail orientation, none of the existing techniques allow for orientation along the dorso-ventral body axis. Here, we present the design of a simple and robust method for passively orienting worms into lateral body positions in microfluidic devices to facilitate inspection of morphological features with specific dorso-ventral alignments. Using this technique, we can position animals into lateral orientations with up to 84% efficiency, compared to 21% using existing methods. We isolated six mutants with neuronal development or neurodegenerative defects, showing that our technology can be used for on-chip analysis and high-throughput visual screens.

Microfluidic Platforms for Quantitative Biology Studies in Model Organisms

“…The embryo array utilizes hydrodynamic focusing to passively and rapidly position embryos in the vertical orientation necessary for the observation of dorsal/ventral developmental morphogen gradients [65]. There have also been microfluidic platforms designed to rapidly manipulate C. elegans into the field of view in a manner that results in preferential orientation of the organism for observation of key neurons or nerve cord puncta [93]. An application of fluorescence imaging that has been extensively used in C. elegans is calcium imaging.…”

“…1.6). Through the control provided by on-chip valves and predictable fluid streams, microfluidic devices enable the isolation of specimens of interest by routing them to holding chambers or outlets [31,50,54,93,[98][99][100]. Sorting is essential in experiments that require the isolation and further processing of organisms with special characteristics.…”

A perspective on optical developments in microfluidic platforms for Caenorhabditis elegans research

“…For instance, using microfluidics to streamline imaging and perform genetic screens has allowed reducing screening time by several orders of magnitude compared to traditional assays where worms are individually and manually picked. 8,[48][49][50] In a chip, thousands of worms can be successively loaded, imaged, phenotyped, and sorted. Generally, fluorescent reporters are used to discriminate worms of different phenotypes ( Fig.…”

“…To address this, technical solutions using suction, 51,52 mechanical restriction by a valve membrane, 53 channel constriction, 54 and CO 2 anesthesia 53 have been demonstrated; a common and reliable microfluidic method is to use valves to trap the worm in a region and use in situ temperature control to cool (to $4 C) and immobilize the worm. 8,[48][49][50] Cooling is fast, reversible, and effective. Compared to traditional immobilization methods that rely primarily on the use of anesthetic drugs, cooling-based immobilization is non-invasive, allows for recovering the worms after imaging and limits adverse developmental effects.…”

“…8,12,49 The first genetic screen of a mutagenized population of C. elegans in a microfluidic device was performed by Crane et al in 2009. 12 More recently in the automated process, novel mutants were identified based on predefined criteria; using machine-learning techniques, the power of using software algorithms can be pushed one step further where the sorting criteria are determined by the software without human intervention.…”