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Microfluidic approaches for immobilizing and positioning C. elegans

C. elegans is a transparent soil-dwelling nematode. The transparent body of the worm allows real-time imaging and analysis of molecular and cellular events in the body. A protein of interest can be fluorescently tagged in vivo and observed. However, screening the dynamic processes in C. elegans requires precise positioning or immobilizing of the worm. Conventionally, the worms were glued or anesthetized that can disrupt the normal functionality of the worm and lead to false results. Moreover, in many cases, it would be very difficult to recover the worm after it has been glued or anesthetized which impedes further screening. Microfluidics technology offers anesthetic-free and glue-free control of the worm inside the chips which allows screening the dynamics and imaging of the worm. Depending on the specifications of the experiment, any of the following approaches can be employed. These designs can be combined with side microchannels to stimulate the worms electrically, mechanically, optically, or chemically while being immobilized. For certain applications, it is required to move the worm to a specific place in the device. The positioning techniques mentioned in the next part can be helpful when designing a positioning tool for a C. elegans study.

Immobilization Methods

The optical transparency of the microfluidic chips along with the high gas permeability and biocompatibility makes them an ideal choice for C. elegans manipulation and immobilization. The worms can simply be released from the chips for further analysis.

Side-Suction: The worms are flown through a straight channel where they are sucked towards the inner wall caused by the side-suction. This method allows for single worm capturing and analysis.

Tapering: This method is suitable for high-throughput screening. Here, worms go through tapered channels were they get stuck in the channel due to back pressure caused by the flow.

Air pressure: Here, the microfluidic chip consists of three layers. The bottom layer is where the worm solution flows through. The top layer is full of air and is used to compress the thin middle layer to immobilize the worm. It has been shown that this method does damage the worms.

CO2 Immobilization: In here, the chip has three layers. The worms run through the bottom layer. CO2 is run through the top layer and protrudes through the thin porous middle layer. Exposure to CO2 immobilizes the worm for 2-3 hours.

 

Side suction Tapering CO2 exposure Air pressure
Chip layers Single-layer Single-layer Two layers Two layers
Throughput Low High Medium Medium
Ease of use Medium Easy Medium Medium
Precision High Medium Medium Medium
Rotation Yes No No No
Damages to the worm Low Low Yes in the long run Yes in the long run

Positioning Techniques

Electrotaxis: C. elegans is responsive to the electric field. It responds to both the direction and the strength of the electric field. This behaviour is called electrotaxis and can be used to move and position the worms inside the channel. Placing the electrodes in the desired place and applying the voltage can help in the voluntarily positioning of the worms.

Chemotaxis: C. elegans is capable of identifying chemical cues via two types of organs located in the head area and the posterior, namely amphids and phasmids. These sensory systems help the nematode to orient itself in the environment. The worm’s response to chemical gradients is helpful for positioning the worms inside microfluidic chips. C. elegans has response mechanisms to various chemicals, odours, and gases that can help the worm identify the food sources. The worm can swim toward or away from a chemical gradient and therefore hold a specific position in the chip.

Phototaxis: C. elegans can respond to light in a process called phototaxis. Although C. elegans does not carry photosensing neurons, it can respond to optical cues via different mechanisms. For example, the blue light can induce acetylcholine in the worm which is a neurotransmitter that causes muscle contraction.

Further Reading

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