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>
	<li>Eastwood, A. L., 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=Tissue+mechanics+govern+the+rapidly+adapting+and+symmetrical+response+to+touch.">Tissue mechanics govern the rapidly adapting and symmetrical response to touch.</a> Proc. Natl. Acad. Sci. 15 (50), E6955-E6963 (2015).</li><li>Katta, S., Krieg, M., Goodman, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeling+Force:+Physical+and+Physiological+Principles+Enabling+Sensory+Mechanotransduction.">Feeling Force: Physical and Physiological Principles Enabling Sensory Mechanotransduction.</a> Annu. Rev. Cell Dev. Biol. 31, 347-371 (2015).</li><li>Krieg, M., Dunn, A. R., Goodman, M. 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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 ...

The overall goal of this technique is to measure how nematodes and their neurons respond to external mechanical stimuli using a microfluidic chip setup with pressure actuators. This method can answer key questions in the fields of mechanobiology and sensory biology such as how cells, tissues and animals respond to mechanical stimulation. The main advantage of this technique is that it sufficiently reduces the worm's mobility in a non-invasive way to allow high-resolution imaging while still having access to the worm's cuticle for mechanical stimulation.This method can also be used to study the influence of mechanical cues on development. It could even be applied to other systems such ex vivo organ exponse or other animals of similar size to C.Elegans. Set up a microscope system for simultaneous excitation of GCaMP and RFP.One option is a discreet light source that transmits only cyan and yellow light. For viewing, use a 10-times objective and high-magnification objective. Also, send the image to a computer via digital camera.Next, include a florescence cube, and if needed, an excitation filter. Add a dichroic mirror to the cube that reflects cyan and yellow light and transmits green and red light. Set up a beam splitter with a long-pass dichroic mirror with a cutoff at 570 nanometers.Also, provide a band-pass emission filter for green light at 525 nanometers with a 50-nanometer width and a band-pass emission filter for red light at 632 nanometers with a 60-nanometer width. Project both beams into the camera's field of view. Make sure the green part is projected to the top half of the screen, and the red part to the bottom.Always use microfluidic chips that are less than one month old. For the setup, first load the gravity flow reservoir with filtered M9.Position the reservoir about 60 centimeters above the chip and connect it to a chip outlet. Next, connect the other outlet to a waste container with two inputs, and connect the second input to the waste container to a peristaltic pump.Make all the connections with polyethylene tubing. Next, assemble interconnects, 50-millimeter tubing lengths with metal tubing connectors on both ends. Press-fit and interconnect onto each of the six actuation fingers and into the worm inlet.Now, position the chip under the optics. Be sure to leave the interconnects attached to the chip as the PDMS will wear out quickly with repeated manipulations. It is vital that the PDMS actuator and the tubing connecting the chip to the air reservoir are properly sealed to ensure good chromatic actuation of the diaphragm.Before loading animals, check for loss of pressure in the microfluidic chip. Measure the deflection of the diaphragm by performing several actuation cycles at the desired pressure. Then, compare the quantitative values with theoretical predictions.If the measured values are not as expected, check for leaks in the tubing or tubing connectors. The PDMS may also have a different elasticity due to an alternative ratio of base polymer and curing agent, or the PD mass could be old and excessively crosslinked. Have prepared age-synchronized-to-young-adult or adult-day-one C.elegans as described by Porta-de-la-Riva and company in their Jove publication from 2012.Now, pick two to five young adults or one-day-old hermaphrodites. Choosing animals of the correct size is critical. Small animals will not get trapped in the channel, and overly large animals will be difficult to remove and can clog the device.Pull the picked worms in a drop of filtered M9.Then, draw them into a length of tubing using a one-milliliter syringe without pulling them into the syringe body itself. Next, connect the tubing to the interconnect at the worm inlet of the chip. Then, activate the gravity flow by opening the valve to the reservoir and starting the pump.Now, observe the trapping channel at four-times magnification and gently push the animals into the device. After loading the animals into the waiting chamber, use the plunger to gently flow one of them to the front of the trapping channel such that its head enters the tapered shape of the channel. If the worm does not fill the entire cross-section of the channel from the nose to near the end of the body, remove the worm.Many times, worms that are too small will go straight through the chip without being trapped. If an animal that got caught in the trapping channel needs to be removed, simply press the plunger of the syringe until the worm disappears from the channel and then load a new worm. Once a worm is properly loaded, switch to florescence mode and increase the magnification.To prevent saturation, be sure to adjust the excitation intensity based on the florescence intensity. Check whether the neurite of a neuron of interest lies across the diaphragm of one of the actuators. That actuator must also be anterior to the cell body of the neuron.If not, adjust the position of the animal by pulling or pushing on the plunger. If that does not help, remove the worm and load a new one. Also, if there happens to be autofluorescence around the neuron, replace the worm.Once a worm is properly loaded, focus on the cell body of the neuron of interest, then determine which actuator the chip is on its anterior side and connect this actuator to the programmable pressure pump using the associated interconnect. If the experiment requires measuring the distance between the actuator and the neuron, fit both into the field of view with the channel wall parallel to the upper and lower edge of the image. Next, define a pressure protocol using the programmable pressure pump.Always start by taking at least 50 images at zero kilopascals to define a baseline. Then, program the stimulus waveform and pressure. Now, run the imaging and pressure protocol.While recording, the neuron of interest must be the brightest spot in a 10-square-pixel area. It cannot move more than 10 pixels in two sequential images, and it must stay in the field of view. While recording, you may observe changes in the florescence intensity when the pressure and the actuators change.This indicates a successful stimulation of the neurons. Between stimuli, include 10 seconds at a constant pressure of zero kilopascal. It is possible to simultaneously record signals from multiple neurons in the field of view so long as they are separated by at least 10 pixels during the entire recording.To keep worms for further study, disconnect the outlets of the chip toward the gravity flow and the waste container. Then, gently press the plunger until the entire worm is pushed through the trapping channel into the flow channel, and continue to press the plunger until the animal appears in a droplet outside of the chip. Now, disconnect the syringe with the tubing and aspirate the worm in the droplet onto an agar plate.To remove and sacrifice a worm in the channel, press the plunger until the entire worm is pushed through the trapping channel and into the flow channel. Then, the worm will flow out of the chip and into the waste container. After setting up the microfluidic chip, the deflection of the diaphragm was tested.The measured values were reproducible with slight variation not exceeding one micron at 450 kilopascal of pressure. After inserting the worms into the inlet of the chip, the skin of a single animal inside the trapping channel was presented to the six actuators. With this design, the PLM neuron usually cannot be immobilized completely, so it free to move laterally and vertically.Although successful activation of PLM neurons is possible, recording calcium transients from them is difficult due to the tail movement. Once in place, the animal was stimulated using one of the six actuators positioned to deliver mechanical stimuli to each of the six TRNs. One of the three different two-second stimuli were presented.A hold at 275 kilopascals, a ramp to 275 kilopascals or a buzz consisting of a 75-kilopascal 10-hertz sine superimposed with the 275-kilopascal step. 10-second gaps with no pressure separated the stimuli. Pressure ramps and pressure steps activated TRNs only if the stimuli achieved pressures above 400 kilopascals, whereas the buzz stimuli robustly activated all TRN.Once mastered, this technique can be performed within five minutes per worm. It is important to remember that a poor seal between the chip and the coverslip, or between the chip and the interconnectors will cause the device to leak and fail. Following this procedure, other methods like voltage imaging or targeted delivery of mechanical stimuli to different neurons can be performed in order to characterize the mechanical response of nociceptors.Also, because the animals can be recovered following this procedure, the technique could be used to screen for mutants defective in their response to mechanical stimuli. Don't forget that working with compressed gases and chemicals can be extremely hazardous. Take precautions such as wearing personal protective garments and working in fume hoods when necessary to avoid those hazards.

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Research

JoVE Journal

Neuroscience

9.7K 조회수.  Stanford University. The overall goal of this technique is to measure how nematodes and their neurons respond to external mechanical stimuli using a microfluidic chip setup with pressure actuators. This method can answer key questions in the fields of mechanobiology and sensory biology such as how cells, tissues and animals respond to mechanical stimulation. The main advantage of this technique is that it sufficiently reduces the worm's mobility in a non-invasive way to allow high-resolution imaging while still h...