An integrated platform enabling optogenetic illumination of Caenorhabditis elegans neurons and muscular force measurement in microstructured environments

Abstract

Optogenetics has been recently applied to manipulate the neural circuits of Caenorhabditis elegans (C. elegans) to investigate its mechanosensation and locomotive behavior, which is a fundamental topic in model biology. In most neuron-related research, free C. elegans moves on an open area such as agar surface. However, this simple environment is different from the soil, in which C. elegans naturally dwells. To bridge up the gap, this paper presents integration of optogenetic illumination of C. elegans neural circuits and muscular force measurement in a structured microfluidic chip mimicking the C. elegans soil habitat. The microfluidic chip is essentially a ∼1 × 1 cm2 elastomeric polydimethylsiloxane micro-pillar array, configured in either form of lattice (LC) or honeycomb (HC) to mimic the environment in which the worm dwells. The integrated system has four key modules for illumination pattern generation, pattern projection, automatic tracking of the worm, and force measurement. Specifically, two optical pathways co-exist in an inverted microscope, including built-in bright-field illumination for worm tracking and pattern generation, and added-in optogenetic illumination for pattern projection onto the worm body segment. The behavior of a freely moving worm in the chip under optogenetic manipulation can be recorded for off-line force measurements. Using wild-type N2 C. elegans, we demonstrated optical illumination of C. elegans neurons by projecting light onto its head/tail segment at 14 Hz refresh frequency. We also measured the force and observed three representative locomotion patterns of forward movement, reversal, and omega turn for LC and HC configurations. Being capable of stimulating or inhibiting worm neurons and simultaneously measuring the thrust force, this enabling platform would offer new insights into the correlation between neurons and locomotive behaviors of the nematode under a complex environment.

Muscle strength deficiency and mitochondrial dysfunction in a muscular dystrophy model of C. elegans and its functional response to drugs

“…Deflections and strength measurements were obtained using standard NemaFlex processing protocol, which involves automated tracking of the deflectable pillars (Fig. 7E) (Ghanbari et al, 2012; Johari et al, 2013; Khare et al, 2015; Qiu et al, 2015). Pillar deflection values extracted during image processing were converted to forces using Timoshenko beam deflection theory (Etheridge et al, 2015; Rahman et al, 2018).…”

Microfluidic Approaches for Manipulating, Imaging, and Screening C. elegans

“…Subsequently, two devices were reported that contained micro-pillar structures for measuring the forces exerted by worms on their microenvironment [ 46 , 47 ]. Johari et al [ 46 ] used PDMS pillar elastic deflection concept to measure the force, and found that despite changes in the placements of pillars in a “honeycomb” or “lattice” configuration, worms continued to move in a sinusoidal pattern but their locomotion speed, undulation frequency, and body force changed significantly.…”

“…Measurement of forces applied on pillars revealed that the mid-body region generates the maximum force. The device by Qiu et al [ 47 ] was designed to expose worms to a particular frequency of light that they tend to avoid and measure force generated during forward or backward movements, or omega turns in the structured environment. A LabVIEW program (National Instruments, Austin, TX, USA) was also developed to assist with image capture and data analysis.…”

Advanced Microfluidic Assays for Caenorhabditis elegans

“…Locomotion. Owing to the precision achievable by microlithographic techniques, researchers have been able to investigate in-depth different locomotion patterns (by varying the size and spacing of posts), applied muscular forces (by measuring the deflection of posts) ( Figure 1D) and motility quantification (time-averaged kinetic power over the swimming cycle) [81][82][83][84][85][86][87][88][89][90]. In contrast to traditional experimental techniques, these systems permitted straightforward dynamic force detection of moving nematodes.…”