using-microfluidics-device-for-mechanical-stimulation-high-resolution

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

One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the ...

Stanford University

New tools for applying force to animals, tissues, and cells are critically needed in order to advance the field of mechanobiology, as few existing tools enable simultaneous imaging of tissue and cell deformation as well as cellular activity in live animals. Here, we introduce a novel microfluidic device that enables high-resolution optical imaging of cellular deformations and activity while applying precise mechanical stimuli to the surface of the worm's cuticle with a pneumatic pressure reservoir. To evaluate device performance, we compared analytical and numerical simulations conducted during the design process to empirical measurements made with fabricated devices. Leveraging the well-characterized touch receptor neurons (TRNs) with an optogenetic calcium indicator as a model mechanoreceptor neuron, we established that individual neurons can be stimulated and that the device can effectively deliver steps as well as more complex stimulus patterns. This microfluidic device is therefore a valuable platform for investigating the mechanobiology of living animals and their mechanosensitive neurons.

Pneumatic stimulation of C. elegans mechanoreceptor neurons in a microfluidic trap.

For decades, Caenorhabditis elegans roundworms have been used to study the sense of touch, and this work has been facilitated by a simple behavioral assay for touch sensation. To perform this classical assay, an experimenter uses an eyebrow hair to gently touch a moving worm and observes whether or not the worm reverses direction. We used two experimental approaches to determine the manner and moment of contact between the eyebrow hair tool and freely moving animals and the forces delivered by the classical assay. Using high-speed video (2500 frames/second), we found that typical stimulus delivery events include a brief moment when the hair is contact with the worm's body and not the agar substrate. To measure the applied forces, we measured forces generated by volunteers mimicking the classical touch assay by touching a calibrated microcantilever. The mean (61 μN) and median forces (26 μN) were more than ten times higher than the 2-μN force known to saturate the probability of evoking a reversal in adult C. elegans. We also considered the eyebrow hairs as an additional source of variation. The stiffness of the sampled eyebrow hairs varied between 0.07 and 0.41 N/m and was correlated with the free length of hair. Collectively, this work establishes that the classical touch assay applies enough force to saturate the probability of evoking reversals in adult C. elegans in spite of its variability among trials and experimenters and that increasing the free length of the hair can decrease the applied force.

Article	Year
Pneumatic stimulation of C. elegans mechanoreceptor neurons in a microfluidic trap. Lab on a chip\| PubMed ID: 28207921	2017
Forces applied during classical touch assays for Caenorhabditis elegans. PloS one\| PubMed ID: 28542494	2017

Adam L. Nekimken

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