Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of <em>C. elegans</em>

Abstract

One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the influence of mechanical stress on cellular function is still poorly understood. In part, this knowledge gap exists because few tools enable simultaneous deformation of tissue and cells, imaging of cellular activity in live animals, and efficient restriction of motility in otherwise highly mobile model organisms, such as the nematode Caenorhabditis elegans. The small size of C. elegans makes them an excellent match to microfluidics-based research devices, and solutions for immobilization have been presented using microfluidic devices. Although these devices allow for high-resolution imaging, the animal is fully encased in polydimethylsiloxane (PDMS) and glass, limiting physical access for delivery of mechanical force or electrophysiological recordings. Recently, we created a device that integrates pneumatic actuators with a trapping design that is compatible with high-resolution fluorescence microscopy. The actuation channel is separated from the worm-trapping channel by a thin PDMS diaphragm. This diaphragm is deflected into the side of a worm by applying pressure from an external source. The device can target individual mechanosensitive neurons. The activation of these neurons is imaged at high-resolution with genetically-encoded calcium indicators. This article presents the general method using C. elegans strains expressing calcium-sensitive activity indicator (GCaMP6s) in their touch receptor neurons (TRNs). The method, however, is not limited to TRNs nor to calcium sensors as a probe, but can be expanded to other mechanically-sensitive cells or sensors.

Microfluidics for mechanobiology of model organisms

“…We designed and published a microfluidic device for mechanical stimulation of worms during high-resolution imaging (Nekimken, Fehlauer, et al, 2017), as well as a detailed protocol for fabricating and operating our device (Fehlauer et al, 2018). The device integrated a trapping channel adapted from a microfluidic platform for studying chemosensation in worms (Chronis, Zimmer, & Bargmann, 2007) with six individually addressable mechanical actuators for stimulating touch receptor neurons.…”

“…The smallest constriction of the trap was approximately half of the worm width, facilitating removal of animals and minimizing the likelihood of clogging. Following on-chip experiments, it was possible to recover organisms, thus enabling long-term studies of individuals (Fehlauer et al, 2018; Hulme et al, 2007; Nekimken, Fehlauer, et al, 2017). …”

“…The process consists of two stages, (1) photolithography of the master mold and (2) replica molding and bonding of PDMS devices. Details can be found in Fehlauer et al (2018) and Nekimken, Fehlauer, et al (2017). Ease of fabrication was an important consideration in the design of the trapping channel and actuators.…”

“…Our microfluidic device also required some external equipment for normal operation, and the detailed instructions can be found in Fehlauer et al (2018). In this section, we describe why we chose to include the additional equipment in our experimental setup.…”

“…To correct for defocusing, we used an additional fluorophore in our intensity measurements. This, in turn, required two excitation wavelengths and a beam splitter for imaging the intensities of both of the fluorophores simultaneously (Fehlauer et al, 2018; Nekimken, Fehlauer, et al, 2017). Different experiments require different optical setups, and the choices made when designing an imaging system can have a major impact on the results of an experiment.…”