Name: using a microfluidics device for mechanical stimulation and high resolution imaging of c. elegans
Uploaded: 2018-02-19T13:00:00
Description: Watch this Scientific Journal Video about Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans at JoVE.com

We thank Sandra N. Manosalvas-Kjono, Purim Ladpli, Farah Memon, Divya Gopisetty, and Veronica Sanchez for support in device design and generation of mutant animals. This research was supported by NIH grants R01EB006745 (to BLP), R01NS092099 (to MBG), K99NS089942 (to MK), F31NS100318 (to ALN) and received funding from the European Research Council (ERC) under the European Union&#39;s Horizon 2020 research and innovation program (grant agreement No. 715243 to MK).

1. Device Fabrication
<ol>
	<li>Download the attached mask file (Supplemental File 1) and generate a chrome mask using a commercial service or in-house facility. As the smallest dimension on the device is 10 &#181;m (actuator membrane thickness), ensure that the mask has sufficiently high resolution, within &#177; 0.25 &#181;m, to reliably produce the features.</li>
	<li>Follow standard SU-8 photolithography methods (e.g., references24,<stron...

<ol>
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B., Chalfie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+DEG/ENaC+protein+MEC-10+regulates+the+transduction+channel+complex+in+Caenorhabditis+elegans+touch+receptor+neurons.">The DEG/ENaC protein MEC-10 regulates the transduction channel complex in Caenorhabditis elegans touch receptor neurons.</a> J. Neurosci. 31 (35), 12695-12704 (2011).</li><li>Lockhead, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tubulin+repertoire+of+Caenorhabditis+elegans+sensory+neurons+and+its+context-dependent+role+in+process+outgrowth.">The tubulin repertoire of Caenorhabditis elegans sensory neurons and its context-dependent role in process outgrowth.</a> Mol. Biol. Cell. 27 (23), 3717-3728 (2016).</li><li>Goodman, M. B., Ernstrom, G. G., Chelur, D. S., O'hagan, R., Yao, C. 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This protocol demonstrates a method for delivering precise mechanical stimulation to the skin of a roundworm trapped in a microfluidic chip. It is intended to facilitate the integration of physical stimuli for answering biological questions and aims to streamline mechanobiology research in biological labs. This method extends previous assays to assess the function of mechanosensory neurons in C. elegans. Previous quantitative and semi-quantitative techniques measured forces1,<s...

The sense of touch provides animals with crucial information about their environment. Depending on the applied force, touch is perceived as innocuous, pleasurable, or painful. The tissue deformation during touch is detected by specialized mechanoreceptor cells embedded in the skin that express receptor proteins, most commonly ion channels. The steps linking force perception to ion channel activation during touch and pain are not fully understood. Even less is known about how the skin tissue filters mechanical deformation and whether mechanoreceptors detect changes in strain or stress1,2,3. This gap in understanding arises, in part, from a lack of suitable tools to apply precise mechanical stimulations to the surface of the skin of a living animal while observing the responses at the cellular level. Whereas atomic force microscopy has been used extensively to apply and measure forces in isolated cells4,5 and also to activate Piezo1 receptors in living cells6, similar experiments using living animals, especially C. elegans, have been notoriously challenging due to the intrinsic mobility of the subject. This challenge is traditionally circumvented by using veterinary- or surgical-grade cyanoacrylate glue to immobilize individual animals on agar pads1,7,8,9. This approach has been productive, but has limitations related to the skill required for immobilization by gluing and the soft agar surface on mechanical compliance. A microfluidics strategy is a complimentary alternative that avoids some of the complications linked to gluing.
The nematode C. elegans is a genetic model organism with a completely mapped nervous system that, due to the animal&#39;s size, is a good fit for microfluidics technology. Microfluidics-based devices offer the advantage that the otherwise extremely mobile animals can be restrained while performing high-resolution imaging and delivery of relevant neuro-modulatory stimuli. With the help of microfluidic technologies, living animals can be immobilized without harm10,11, enabling monitoring of behavioral activity over the entire lifetime12,13 and high-resolution imaging of neuronal activity14,15,16,17. Further, many mechanoreceptor neurons needed for the sense of touch and pain can be characterized on their physiological1,8, mechanical4,18,19, and molecular level20,21,22.
C. elegans senses gentle mechanical stimuli to its body wall using six TRNs, three of which innervate the animal&#39;s anterior (ALML/R and AVM) and three of which innervate the animal&#39;s posterior (PLML/R and PVM). The ion channel molecules needed for transducing an applied force into a biochemical signal have been extensively studied in its TRNs8. This article presents a microfluidic platform23 that enables researchers to apply precise mechanical forces to the skin of an immobilized C. elegans roundworm, while reading out the deformation of its internal tissues by optical imaging. In addition to presenting well-defined mechanical stimuli, calcium transients can be recorded in mechanoreceptor neurons with subcellular resolution and correlated with morphological and anatomical features. The device consists of a central trapping channel that holds a single animal and presents its skin next to six pneumatic actuation channels (Figure 1 and Figure 2). The six channels are positioned along the trapping channel to deliver mechanical stimuli to each of the worm&#39;s six TRNs. These channels are separated from the trapping chamber by thin PDMS diaphragms, which can be driven by an external air pressure source (Figure 1). We calibrated the deflection with respect to pressure and provide the measurements in this article. Each actuator can be addressed individually and used to stimulate a mechanoreceptor of choice. The pressure is delivered using a piezo-driven pressure pump but any alternative device can be used. We show that the pressure protocol can be used to activate TRNs in vivo and demonstrate operating devices suitable for delivering mechanical stimuli to adult C. elegans, loading adult animals into devices, performing calcium imaging experiments, and analyzing the results. Device fabrication consists of two main steps: 1) photolithography to make a mold from SU-8; and 2) molding PDMS to make a device. For the sake of brevity and clarity, readers are referred to previously published articles and protocols24,25 for instructions on how to produce the molds and devices.

<table>
	<tbody>
		<tr>
			<td>Chrome mask</td>
			<td>Compugraphics (http://www.compugraphics-photomasks.com/)</td>
			<td></td>
			<td>5&#39;&#39;, designed in AutoCAD (Autodesk, Inc.)</td>
		</tr>
		<tr>
			<td>Chrome mask</td>
			<td>Mitani-Micronics (http://www.mitani-micro.co.jp/en/)</td>
			<td></td>
			<td>5&#39;&#39;, designed in AutoCAD (Autodesk, Inc.)</td>
		</tr>
		<tr>
			<td>Chrome mask</td>
			<td>Kuroda-Electric (http://www.kuroda-electric.eu/</td>
			<td></td>
			<td>5&#39;&#39;, designed in AutoCAD (Autodesk, Inc.)</td>
		</tr>
		<tr>
			<td>4&#39;&#39; Silicon wafer (B-test)</td>
			<td>Stanford Nanofabrication Facility</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 2002</td>
			<td>MicroChem</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 2050</td>
			<td>MicroChem</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spin-coater</td>
			<td>Laurell Technologies</td>
			<td>WS-400BZ-6NPP/LITE</td>
			<td></td>
		</tr>
		<tr>
			<td>Exposure timer</td>
			<td>Optical Associates, Inc</td>
			<td>OAI 150</td>
			<td></td>
		</tr>
		<tr>
			<td>Illumination controller</td>
			<td>Optical Associates, Inc</td>
			<td>2105C2</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 developer</td>
			<td>MicroChem</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2-Propanol</td>
			<td>Fisher Scientific</td>
			<td>A426F-1GAL</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Fisher Scientific</td>
			<td>A18-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichloromethylsilane (TCMS)</td>
			<td>Sigma-Aldrich</td>
			<td>92361-500ML</td>
			<td>Caution: TCMS is toxic and water-reactive</td>
		</tr>
		<tr>
			<td>Sylgard 184 Elastomer Kit</td>
			<td>Dow Corning</td>
			<td></td>
			<td>PDMS prepolymer</td>
		</tr>
		<tr>
			<td>Biopsy punch, 1 mm</td>
			<td>VWR</td>
			<td>95039-090</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen Plasma Asher</td>
			<td>Branson/IPC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Small metal tubing (0.635 mm OD, 0.4318 mm ID, 12.7 mm long); gage size 23TW</td>
			<td>New England Small Tube Corporation</td>
			<td>NE-1300-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene syringe filter, 0.22 &#956;m</td>
			<td>Thermo Scientific</td>
			<td>725-2520</td>
			<td>to filter all solution, small particles would clog the chip</td>
		</tr>
		<tr>
			<td>Polyethylene tubing; 0.9652 mm OD, 0.5842 mm ID</td>
			<td>Solomon Scientific</td>
			<td>BPE-T50</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe, 1 ml</td>
			<td>BD Scientific</td>
			<td>309628</td>
			<td>for worm trapping and release</td>
		</tr>
		<tr>
			<td>Syringe, 20 ml</td>
			<td>BD Scientific</td>
			<td>309661</td>
			<td>for gravity-based flow</td>
		</tr>
		<tr>
			<td>Gilson Minipuls 3, Peristaltic pump</td>
			<td>Gilson</td>
			<td></td>
			<td>to suck solutions and worms out of the chip</td>
		</tr>
		<tr>
			<td>Microfluidic flow controller, equipped with 0&#8211;800 kPa pressure channel</td>
			<td>Elveflow</td>
			<td>OB1 MK3</td>
			<td>pressure delivery</td>
		</tr>
		<tr>
			<td>Water-Resistant Clear Poly- urethane Tubing, 4 mm ID and 6 mm OD</td>
			<td>McMaster-Carr</td>
			<td>5195 T52</td>
			<td>connection from house air to pressure pump</td>
		</tr>
		<tr>
			<td>Water-Resistant Clear Polyurethane Tubing, 2.6mm ID and 4mm OD</td>
			<td>McMaster-Carr</td>
			<td>5195 T51</td>
			<td>connect pressure pump to small tubng</td>
		</tr>
		<tr>
			<td>Push-to-Connect Tube Fitting for Air</td>
			<td>McMaster-Carr</td>
			<td>5111K468</td>
			<td>metric - imperial converter</td>
		</tr>
		<tr>
			<td>Straight Connector for 6 mm &#215; 1/4&#8243; Tube OD</td>
			<td>McMaster-Carr</td>
			<td>5779 K258</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica DMI 4000 B microscopy system</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>63&#215;/1.32 NA HCX PL APO oil objective</td>
			<td>Leica</td>
			<td>506081</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamamatsu Orca-Flash 4.0LT digital CMOS camera</td>
			<td>Hamamatsu</td>
			<td>C11440-42U</td>
			<td></td>
		</tr>
		<tr>
			<td>Lumencor Spectra X light engine</td>
			<td>Lumencor</td>
			<td></td>
			<td>With cyan and green/yellow light source</td>
		</tr>
		<tr>
			<td>Excitation beam splitter</td>
			<td>Chroma</td>
			<td>59022bs</td>
			<td>in the microscope</td>
		</tr>
		<tr>
			<td>Hamamatsu W-view Gemini Image splitting optics</td>
			<td>Hamamatsu</td>
			<td>A12801-01</td>
			<td>to split green and red emission and project them on different areas on the camera chip</td>
		</tr>
		<tr>
			<td>Emission beam splitter</td>
			<td>Chroma</td>
			<td>T570lpxr</td>
			<td>in the image splitter</td>
		</tr>
		<tr>
			<td>Emission filters GCamp6s</td>
			<td>Chroma</td>
			<td>ET525/50m</td>
			<td>in the image splitter</td>
		</tr>
		<tr>
			<td>Emission filters mCherry</td>
			<td>Chroma</td>
			<td>ET632/60m</td>
			<td>in the image splitter</td>
		</tr>
	</tbody>
</table>

using a microfluidics device for mechanical stimulation and high resolution imaging of c. elegans

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.

SU-8 Lithography and Chip Bonding 
The lithography protocol and PDMS molding follow standard procedures. Details can be found elsewhere23,24,25,26. The PDMS should peel off the wafer without problems after curing. If the SU-8 features rip off during PDMS peeling, either the SU-8 adhesion layer or the silanization was insufficient. If plasma...

Watch this Scientific Journal Video about Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans at JoVE.com

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

New tools for mechanobiology research are needed to understand how mechanical stress activates biochemical pathways and elicits biological responses. Here, we showcase a new method for selective mechanical stimulation of immobilized animals with a microfluidic trap allowing high-resolution imaging of cellular responses.

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

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

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