Continuous-flow C. elegans fluorescence expression analysis with real-time image processing through microfluidics.

The nematode Caenorhabditis elegans has become an essential model organism in neuroscience research because of its stereotyped anatomy, relevance to human biology, and capacity for genetic manipulation. To solve the intrinsic challenges associated with performing manual operations on C. elegans, many automated chip designs based on immobilization-imaging-release approaches have been proposed. These designs are prone to limitations such as the exertion of physical stress on the worms and limited throughput. In this work, a continuous-flow, high-throughput, automated C. elegans analyzer based on droplet encapsulation and real-time image processing was developed to analyze fluorescence expression in worms. To demonstrate its capabilities, two strains of C. elegans nematodes with different levels of expression of green fluorescent protein (GFP) were first mixed in a buffer solution. The worms were encapsulated in water-in-oil droplets to restrict random locomotion. The droplets were closely packed in a two-layer polydimethylsiloxane (PDMS) platform and were flowed through a narrow straight channel, in which a region of interest (ROI) was defined and continuously recorded by a frame acquisition device. Based on the number of pixels counted in the selected color range, our custom software analyzed GFP expression to differentiate between two strains with nearly 100% accuracy and a throughput of 0.5 seconds/worm.

A continuous-flow C. elegans sorting system with integrated optical fiber detection and laminar flow switching.

We present a high-throughput continuous-flow C. elegans sorting device that works based on integrated optical fiber detection and laminar flow switching. Two types of genetically engineered nematodes are allowed to flow into the device and their genotypes are detected based on their fluorescence, without the need for immobilization, by integrated optical fibers. A novel dynamic fluidic switch sorts the nematodes to desired outlets. By changing input pressures of the control inlets, the laminar flow path is altered to steer the nematodes to appropriate outlets. Compared to previously reported microfluidic C. elegans sorting devices, sorting in this system is conducted in a continuous flow environment without any immobilization technique or need for multilayer mechanical valves to open and close the outlets. The continuous flow sorter not only increases the throughput but also avoids any kind of invasive or possibly damaging mechanical or chemical stimulus. We have characterized both the detection and the switching accuracy of the sorting device at different flow rates, and efficiencies approaching 100% can be achieved with a high throughput of about one nematode per second. To confirm that there was no significant damage to C. elegans following sorting, we recovered the sorted worms, finding no deaths and no differences in behavior and propagation compared to control.