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).

<ol><li>Eastwood, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue+mechanics+govern+the+rapidly+adapting+and+symmetrical+response+to+touch%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci%22%5BJournal%5D)">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/pubmed?term=((Feeling+Force%3A+Physical+and+Physiological+Principles+Enabling+Sensory+Mechanotransduction%5BTitle%5D)+AND+%22Annu.+Rev.+Cell+Dev.+Biol%22%5BJournal%5D)">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. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanical+systems+biology+of+C.+elegans+touch+sensation%5BTitle%5D)+AND+%22BioEssays%22%5BJournal%5D)">Mechanical systems biology of C. elegans touch sensation.</a> BioEssays. 37 (3), 335-344 (2015).</li>
<li>Krieg, M., Dunn, A. R., Goodman, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanical+control+of+the+sense+of+touch+by+%CE%B2-spectrin%5BTitle%5D)+AND+%22Nat.+Cell+Biol%22%5BJournal%5D)">Mechanical control of the sense of touch by β-spectrin.</a> Nat. Cell Biol. 16 (3), 224-233 (2014).</li>
<li>Krieg, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tensile+forces+govern+germ-layer+organization+in+zebrafish%5BTitle%5D)+AND+%22Nat+Cell+Biol%22%5BJournal%5D)">Tensile forces govern germ-layer organization in zebrafish.</a> Nat Cell Biol. 10 (4), 429-436 (2008).</li>
<li>Gaub, B. M., Müller, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanical+stimulation+of+Piezo1+receptors+depends+on+extracellular+matrix+proteins+and+directionality+of+force%5BTitle%5D)+AND+%22Nano+Lett%22%5BJournal%5D)">Mechanical stimulation of Piezo1 receptors depends on extracellular matrix proteins and directionality of force.</a> Nano Lett. 17 (3), 2064-2072 (2017).</li>
<li>Geffeney, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DEG%2FENaC+but+not+TRP+channels+are+the+major+mechanoelectrical+transduction+channels+in+a+c.+Elegans+nociceptor%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">DEG/ENaC but not TRP channels are the major mechanoelectrical transduction channels in a c. Elegans nociceptor.</a> Neuron. 71 (5), 845-857 (2011).</li>
<li>O'Hagan, R., Chalfie, M., Goodman, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+MEC-4+DEG%2FENaC+channel+of+Caenorhabditis+elegans+touch+receptor+neurons+transduces+mechanical+signals%5BTitle%5D)+AND+%22Nat.+Neurosci%22%5BJournal%5D)">The MEC-4 DEG/ENaC channel of Caenorhabditis elegans touch receptor neurons transduces mechanical signals.</a> Nat. Neurosci. 8 (1), 43-50 (2005).</li>
<li>Suzuki, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+Vivo+Imaging+of+C.+elegans+Mechanosensory+Neurons+Demonstrates+a+Specific+Role+for+the+MEC-4+Channel+in+the+Process+of+Gentle+Touch+Sensation%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">In Vivo Imaging of C. elegans Mechanosensory Neurons Demonstrates a Specific Role for the MEC-4 Channel in the Process of Gentle Touch Sensation.</a> Neuron. 39 (6), 1005-1017 (2003).</li>
<li>Kopito, R. B., Levine, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Durable+spatiotemporal+surveillance+of+Caenorhabditis+elegans+response+to+environmental+cues%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">Durable spatiotemporal surveillance of Caenorhabditis elegans response to environmental cues.</a> Lab Chip. 14 (4), 764-770 (2014).</li>
<li>Chokshi, T. V., Ben-Yakar, A., Chronis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CO2+and+compressive+immobilization+of+C.+elegans+on-chip%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">CO2 and compressive immobilization of C. elegans on-chip.</a> Lab Chip. 9 (1), 151(2009).</li>
<li>Hulme, S. E., Shevkoplyas, S. S., McGuigan, A. P., Apfeld, J., Fontana, W., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lifespan-on-a-chip%3A+microfluidic+chambers+for+performing+lifelong+observation+of+C.+elegans%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">Lifespan-on-a-chip: microfluidic chambers for performing lifelong observation of C. elegans.</a> Lab Chip. 10 (5), 589-597 (2010).</li>
<li>Li, S., Stone, H. a, Murphy, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+microfluidic+device+and+automatic+counting+system+for+the+study+of+C.+elegans+reproductive+aging%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">A microfluidic device and automatic counting system for the study of C. elegans reproductive aging.</a> Lab Chip. 15 (2), 524-531 (2015).</li>
<li>Chokshi, T. V., Bazopoulou, D., Chronis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+automated+microfluidic+platform+for+calcium+imaging+of+chemosensory+neurons+in+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">An automated microfluidic platform for calcium imaging of chemosensory neurons in Caenorhabditis elegans.</a> Lab Chip. 10 (20), 2758-2763 (2010).</li>
<li>Mishra, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Using+microfluidics+chips+for+live+imaging+and+study+of+injury+responses+in+Drosophila+larvae%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Using microfluidics chips for live imaging and study of injury responses in Drosophila larvae.</a> J. Vis. Exp. , e50998(2014).</li>
<li>Chronis, N., Zimmer, M., Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microfluidics+for+in+vivo+imaging+of+neuronal+and+behavioral+activity+in+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Nat.+Methods%22%5BJournal%5D)">Microfluidics for in vivo imaging of neuronal and behavioral activity in Caenorhabditis elegans.</a> Nat. Methods. 4 (9), 727-731 (2007).</li>
<li>Krajniak, J., Lu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-term+high-resolution+imaging+and+culture+of+C.+elegans+in+chip-gel+hybrid+microfluidic+device+for+developmental+studies%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">Long-term high-resolution imaging and culture of C. elegans in chip-gel hybrid microfluidic device for developmental studies.</a> Lab Chip. 10 (14), 1862-1868 (2010).</li>
<li>Vasquez, V., Krieg, M., Lockhead, D., Goodman, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phospholipids+that+Contain+Polyunsaturated+Fatty+Acids+Enhance+Neuronal+Cell+Mechanics+and+Touch+Sensation%5BTitle%5D)+AND+%22CellReports%22%5BJournal%5D)">Phospholipids that Contain Polyunsaturated Fatty Acids Enhance Neuronal Cell Mechanics and Touch Sensation.</a> CellReports. 6 (1), 70-80 (2013).</li>
<li>Krieg, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+defects+in+%CE%B2-spectrin+and+tau+sensitize+C.+elegans+axons+to+movement-induced+damage+via+torque-tension+coupling%5BTitle%5D)+AND+%22Elife%22%5BJournal%5D)">Genetic defects in β-spectrin and tau sensitize C. elegans axons to movement-induced damage via torque-tension coupling.</a> Elife. 6 (2010), e20172(2017).</li>
<li>Arnadóttir, J., O'Hagan, R., Chen, Y., Goodman, M. B., Chalfie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+DEG%2FENaC+protein+MEC-10+regulates+the+transduction+channel+complex+in+Caenorhabditis+elegans+touch+receptor+neurons%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">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="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5170555/">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. A., Chalfie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MEC-2+regulates+C.+elegans+DEG%2FENaC+channels+needed+for+mechanosensation%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">MEC-2 regulates C. elegans DEG/ENaC channels needed for mechanosensation.</a> Nature. 415 (6875), 1039-1042 (2002).</li>
<li>Nekimken, A., Fehlauer, H., Kim, A., Goodman, M., Pruitt, B. L., Krieg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pneumatic+stimulation+of+C.+elegans+mechanoreceptor+neurons+in+a+microfluidic+trap%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">Pneumatic stimulation of C. elegans mechanoreceptor neurons in a microfluidic trap.</a> Lab Chip. , (2017).</li>
<li>Brower, K., White, A. K., Fordyce, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multi-step+Variable+Height+Photolithography+for+Valved+Multilayer+Microfluidic+Devices%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices.</a> J. Vis. Exp. (119), e55276(2017).</li>
<li>Jenkins, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+prototyping+of+PDMS+devices+using+SU-8+lithography%5BTitle%5D)+AND+%22Methods+Mol.+Biol%22%5BJournal%5D)">Rapid prototyping of PDMS devices using SU-8 lithography.</a> Methods Mol. Biol. 949 (1), 153-168 (2013).</li>
<li>Faustino, V., Catarino, S. O., Lima, R., Minas, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biomedical+microfluidic+devices+by+using+low-cost+fabrication+techniques%3A+A+review%5BTitle%5D)+AND+%22J.+Biomech%22%5BJournal%5D)">Biomedical microfluidic devices by using low-cost fabrication techniques: A review.</a> J. Biomech. 49 (11), 2280-2292 (2016).</li>
<li>Xia, Y., Whitesides, G. M. <a target="_blank" href="https://www.fkit.unizg.hr/_download/repository/Soft_litography_Whitesides_Review__aciee98.pdf">SOFT LITHOGRAPHY.</a> Annu. Rev. Mater. Sci. 28 (1), 153-184 (1998).</li>
<li>Chen, T. -W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ultrasensitive+fluorescent+proteins+for+imaging+neuronal+activity%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Ultrasensitive fluorescent proteins for imaging neuronal activity.</a> Nature. 499 (7458), 295-300 (2013).</li>
<li>Cho, Y., Porto, D., Hwang, H., Grundy, L., Schafer, W. R., Lu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Automated+and+controlled+mechanical+stimulation+and+functional+imaging+in+vivo+in+C.+elegans%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">Automated and controlled mechanical stimulation and functional imaging in vivo in C. elegans.</a> Lab Chip. , (2017).</li>
<li>Edwards, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+molecular+solution+for+ultraviolet+light+detection+in+Caenorhabditis+elegans%5BTitle%5D)+AND+%22PLoS+Biol%22%5BJournal%5D)">A novel molecular solution for ultraviolet light detection in Caenorhabditis elegans.</a> PLoS Biol. 6 (8), 1715-1729 (2008).</li>
<li>Porta-de-la-Riva, M., Fontrodona, L., Villanueva, A., Cerón, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Basic+Caenorhabditis+elegans+methods%3A+synchronization+and+observation%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Basic Caenorhabditis elegans methods: synchronization and observation.</a> J. Vis. Exp. (64), e4019(2012).</li>
<li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fiji%3A+an+open-source+platform+for+biological-image+analysis%5BTitle%5D)+AND+%22Nat.+Methods%22%5BJournal%5D)">Fiji: an open-source platform for biological-image analysis.</a> Nat. Methods. 9 (7), 676-682 (2012).</li>
<li>Cho, Y., Porto, D. A., Hwang, H., Grundy, L. J. <a target="_blank" href="http://www.biorxiv.org/content/early/2017/02/10/107318">High-Throughput Controlled Mechanical Stimulation and Functional Imaging In Vivo.</a> BiorXiv. , (2017).</li>
<li>Petzold, B. C., Park, S. -J., Mazzochette, E. A., Goodman, M. B., Pruitt, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MEMS-based+force-clamp+analysis+of+the+role+of+body+stiffness+in+C.+elegans+touch+sensation%5BTitle%5D)+AND+%22Integr.+Biol.+%28Camb%29%22%5BJournal%5D)">MEMS-based force-clamp analysis of the role of body stiffness in C. elegans touch sensation.</a> Integr. Biol. (Camb). 5 (6), 853-864 (2013).</li>
<li>Nekimken, A. L., Mazzochette, E. A., Goodman, M. B., Pruitt, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Forces+applied+during+classical+touch+assays+for+Caenorhabditis+elegans%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Forces applied during classical touch assays for Caenorhabditis elegans.</a> PLoS One. 12 (5), e0178080(2017).</li>
<li>Chronis, N., Zimmer, M., Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microfluidics+for+in+vivo+imaging+of+neuronal+and+behavioral+activity+in+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Nat.+Methods%22%5BJournal%5D)">Microfluidics for in vivo imaging of neuronal and behavioral activity in Caenorhabditis elegans.</a> Nat. Methods. 4 (9), 727-731 (2007).</li>
<li>Gilleland, C. L., Rohde, C. B., Zeng, F., Yanik, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microfluidic+immobilization+of+physiologically+active+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Nat.+Protoc%22%5BJournal%5D)">Microfluidic immobilization of physiologically active Caenorhabditis elegans.</a> Nat. Protoc. 5 (12), 1888-1902 (2010).</li>
<li>Chuang, H. -S., Raizen, D. M., Lamb, A., Dabbish, N., Bau, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dielectrophoresis+of+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">Dielectrophoresis of Caenorhabditis elegans.</a> Lab Chip. 11 (4), 599(2011).</li>
<li>Christensen, A. M., Chang-Yen, D. A., Gale, B. K. <a target="_blank" href="http://iopscience.iop.org/article/10.1088/0960-1317/15/5/005">Characterization of interconnects used in PDMS microfluidic systems.</a> J. Micromechanics Microengineering. 15 (5), 928-934 (2005).</li>
<li>Gilpin, W., Uppaluri, S., Brangwynne, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Worms+under+Pressure%3A+Bulk+Mechanical+Properties+of+C.+elegans+Are+Independent+of+the+Cuticle%5BTitle%5D)+AND+%22Biophys.+J%22%5BJournal%5D)">Worms under Pressure: Bulk Mechanical Properties of C. elegans Are Independent of the Cuticle.</a> Biophys. J. 108 (8), 1887-1898 (2015).</li>
</ol>

The authors have nothing to disclose.

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&#039;&#039;, 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&#039;&#039;, 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&#039;&#039;, designed in AutoCAD (Autodesk, Inc.)</td></tr><tr><td>4&#039;&#039; 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 &amp;mu;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&amp;ndash;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 &amp;times; 1/4&amp;Prime; 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&amp;times;/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 ...

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

Research

JoVE Journal

Neuroscience

10.6K Views.  Stanford University. Nieuwe hulpmiddelen voor het mechanobiology onderzoek nodig zijn om te begrijpen hoe mechanische spanning activeert biochemische paden en lokt biologische reacties. Hier presenteren we een nieuwe methode voor selectief mechanische stimulatie van geïmmobiliseerdet dieren met een val van de microfluidic waardoor hoge resolutie beeldvorming van cellulaire reacties.

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

Assaying &beta;-amyloid Toxicity using a Transgenic C. elegans Model

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant mechanism(s) of toxicity are still unclear. A&beta; is also deposited intramuscularly in Inclusion Body Myositis, a severe human myopathy. The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express human A&beta;. Depending on the tissue or timing of A&beta; expression, transgenic worms can have readily measurable phenotypes that serve as a read-out of A&beta; toxicity. For example, transgenic worms with pan-neuronal A&beta; expression have defects is associative learning (Dosanjh et al. 2009), while transgenic worms with constitutive muscle-specific expression show a progressive, age-dependent paralysis phenotype (Link, 1995; Cohen et al. 2006). One particularly useful C. elegans model employs a temperature-sensitive mutation in the mRNA surveillance system to engineer temperature-inducible muscle expression of an A&beta; transgene, resulting in a reproducible paralysis phenotype upon temperature upshift (Link et al. 2003). Treatments that counter A&beta; toxicity in this model [e.g., expression of a protective transgene (Hassan et al. 2009) or exposure to Ginkgo biloba extracts (Wu et al. 2006)] reproducibly alter the rate of paralysis induced by temperature upshift of these transgenic worms. Here we describe our protocol for measuring the rate of paralysis in this transgenic C. elegans model, with particular attention to experimental variables that can influence this measurement.

Assaying &#946;-amyloid Toxicity using a Transgenic C. elegans Model

The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express the human &beta;-amyloid peptide (A&beta;). Induced expression of A&beta; in C. elegans muscle leads to a rapid, reproducible paralysis phenotype that can be used to monitor treatments that modulate A&beta; toxicity.

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant ...

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

Bioengineering

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and pathways as well as its ease of cultivation. Several C. elegans disease models have been reported, including neurodegenerative disorders such as Parkinson's disease (PD), which involves the degeneration of dopaminergic (DA) neurons 1. Both transgenes and neurotoxic chemicals have been used to induce DA neurodegeneration and consequent movement defects in worms, allowing for investigations into the basis of neurodegeneration and screens for neuroprotective genes and compounds 2,3.
Screens in lower eukaryotes like C. elegans provide an efficient and economical means to identify compounds and genes affecting neuronal signaling. Conventional screens are typically performed manually and scored by visual inspection; consequently, they are time-consuming and prone to human errors. Additionally, most focus on cellular level analysis while ignoring locomotion, which is an especially important parameter for movement disorders.
We have developed a novel microfluidic screening system (Figure 1) that controls and quantifies C. elegans' locomotion using electric field stimuli inside microchannels. We have shown that a Direct Current (DC) field can robustly induce on-demand locomotion towards the cathode ("electrotaxis") 4. Reversing the field's polarity causes the worm to quickly reverse its direction as well. We have also shown that defects in dopaminergic and other sensory neurons alter the swimming response 5. Therefore, abnormalities in neuronal signaling can be determined using locomotion as a read-out. The movement response can be accurately quantified using a range of parameters such as swimming speed, body bending frequency and reversal time.
Our work has revealed that the electrotactic response varies with age. Specifically, young adults respond to a lower range of electric fields and move faster compared to larvae 4. These findings led us to design a new microfluidic device to passively sort worms by age and phenotype 6.
We have also tested the response of worms to pulsed DC and Alternating Current (AC) electric fields. Pulsed DC fields of various duty cycles effectively generated electrotaxis in both C. elegans and its cousin C. briggsae 7. In another experiment, symmetrical AC fields with frequencies ranging from 1 Hz to 3 KHz immobilized worms inside the channel 8.
Implementation of the electric field in a microfluidic environment enables rapid and automated execution of the electrotaxis assay. This approach promises to facilitate high-throughput genetic and chemical screens for factors affecting neuronal function and viability.

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

A semi-automated micro-electro-fluidic method to induce on-demand locomotion in Caenorhabditis elegans is described. This method is based on the neurophysiologic phenomenon of worms responding to mild electric fields (&#x201C;electrotaxis&#x201D;) inside microfluidic channels. Microfluidic electrotaxis serves as a rapid, sensitive, low-cost, and scalable technique to screen for factors affecting neuronal health.

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and ...

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved useful as a convenient live imaging platform providing capabilities for precise control of experimental conditions in real time. In this article, we demonstrate live imaging of individual worms employing WormSpa, a previously-published custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes. To illustrate the capability, we performed proof-of-principle experiments in which worms were infected in the device with pathogenic bacteria, and the dynamics of expression of immune response genes and egg laying were monitored continuously in individual animals. The simple design and operation of this device make it suitable for users with no previous experience with microfluidic-based experiments. We propose that this approach will be useful for many researchers interested in longitudinal observations of biological processes under well-defined conditions.

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In this article, we demonstrate live imaging of individual worms employing a custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes.

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved ...

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means of identifying radiosensitive sites in living organisms. This paper describes a series of on-chip immobilization methods developed for the targeted microbeam irradiation of live individuals of Caenorhabditis elegans. Notably, the treatment of the polydimethylsiloxane (PDMS) microfluidic chips that we previously developed to immobilize C. elegans individuals without the need for anesthesia is explained in detail. This chip, referred to as a worm sheet, is resilient to allow the microfluidic channels to be expanded, and the elasticity allows animals to be enveloped gently. Also, owing to the self-adsorption capacity of the PDMS, animals can be sealed in the channels by covering the surface of the worm sheet with a thin cover film, in which animals are not pushed into the channels for enclosure. By turning the cover film over, we can easily collect the animals. Furthermore, the worm sheet shows water retention and allows C. elegans individuals to be subjected to microscopic observation for long periods under live conditions. In addition, the sheet is only 300 &#181;m thick, allowing heavy ions such as carbon ions to pass through the sheet enclosing the animals, thus allowing the ion particles to be detected and the applied radiation dose to be measured accurately. Because selection of the cover films used for enclosing the animals is very important for successful long-term immobilization, we conducted the selection of the suitable cover films and showed a recommended one among some films. As an application example of the chip, we introduced imaging observation of muscular activities of animals enclosing the microfluidic channel of the worm sheet, as well as the microbeam irradiation. These examples indicate that the worm sheets have greatly expanded the possibilities for biological experiments.

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

A series of immobilization methods has been established to allow the targeted irradiation of live Caenorhabditis elegans individuals using a recently developed ultra-thin polydimethylsiloxane microfluidic chip with water retention. This novel on-chip immobilization is also adequate for imaging observations. The detailed treatment and application examples of the chip are explained.

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means ...

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

The use of calcium indicators has greatly enhanced our understanding of neural dynamics and regulation. The nematode Caenorhabditis elegans, with its completely mapped nervous system and transparent anatomy, presents an ideal model for understanding real-time neural dynamics using calcium indicators. In combination with microfluidic technologies and experimental designs, calcium-imaging studies using these indicators are performed in both free-moving and trapped animals. However, most previous studies utilizing trapping devices, such as the olfactory chip described in Chronis et al., have devices designed for use in the more common hermaphrodite, as the less common male is both morphologically and structurally dissimilar. An adapted olfactory chip was designed and fabricated for increased efficiency in male neuronal imaging with using young adult animals. A turn was incorporated into the worm loading port to rotate the animals and to allow for the separation of the individual neurons within a bilateral pair in 2D imaging. Worms are exposed to a controlled flow of odorant within the microfluidic device, as described in previous hermaphrodite studies. Calcium transients are then analyzed using the open-source software ImageJ. The procedure described herein should allow for an increased amount of male-based C. elegans calcium imaging studies, deepening our understanding of the mechanisms of sex-specific neuronal signaling.

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

The use of an adapted &#34;olfactory chip&#34; for the efficient calcium imaging of C. elegans males is described here. Studies of male exposure to glycerol and a pheromone are also shown.

The use of calcium indicators has greatly enhanced our understanding of neural dynamics and regulation. The nematode Caenorhabditis elegans, with its ...

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding these biological processes often requires long-term and repeated imaging of the same animal. Long recovery times associated with conventional immobilization methods done on agar pads have detrimental effects on animal health making it inappropriate to repeatedly image the same animal over long periods of time. This paper describes a microfluidic chip design, fabrication method, on-chip C. elegans culturing protocol, and three examples of long-term imaging to study developmental processes in individual animals. The chip, fabricated with polydimethylsiloxane and bonded on a cover glass, immobilizes animals on a glass substrate using an elastomeric membrane that is deflected using nitrogen gas. Complete immobilization of C. elegans enables robust time-lapse imaging of cellular and sub-cellular events in an anesthetic-free manner. A channel geometry with a large cross-section allows the animal to move freely within two partially sealed isolation membranes permitting growth in the channel with a continuous food supply. Using this simple chip, imaging of developmental phenomena such as neuronal process growth, vulval development, and dendritic arborization in the PVD sensory neurons, as the animal grows inside the channel, can be performed. The long-term growth and imaging chip operates with a single pressure line, no external valves, inexpensive fluidic consumables, and utilizes standard worm handling protocols that can easily be adapted by other laboratories using C. elegans.

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

The protocol describes a simple microfluidic chip design and microfabrication methodology used to grow C. elegans in presence of a continuous food supply for up to 36 h. The growth and imaging device also enables intermittent long-term high-resolution imaging of cellular and sub-cellular processes during development for several days.

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding ...

Mechanostimulation of Multicellular Organisms Through a High-Throughput Microfluidic Compression System

During embryogenesis, coordinated cell movement generates mechanical forces that regulate gene expression and activity. To study this process, tools such as aspiration or coverslip compression have been used to mechanically stimulate whole embryos. These approaches limit experimental design as they are imprecise, require manual handling, and can process only a couple of embryos simultaneously. Microfluidic systems have great potential for automating such experimental tasks while increasing throughput and precision. This article describes a microfluidic system developed to precisely compress whole Drosophila melanogaster (fruit fly) embryos. This system features microchannels with pneumatically actuated deformable sidewalls and enables embryo alignment, immobilization, compression, and post-stimulation collection. By parallelizing these microchannels into seven lanes, steady or dynamic compression patterns can be applied to hundreds of Drosophila embryos simultaneously. Fabricating this system on a glass coverslip facilitates the simultaneous mechanical stimulation and imaging of samples with high-resolution microscopes. Moreover, the utilization of biocompatible materials, like PDMS, and the ability to flow fluid through the system make this device capable of long-term experiments with media-dependent samples. This approach also eliminates the requirement for manual mounting which mechanically stresses samples. Furthermore, the ability to quickly collect samples from the microchannels enables post-stimulation analyses, including -omics assays which require large sample numbers unattainable using traditional mechanical stimulation approaches. The geometry of this system is readily scalable to different biological systems, enabling numerous fields to benefit from the functional features described herein including high sample throughput, mechanical stimulation or immobilization, and automated alignment.

The present protocol describes the design, fabrication, and characterization of a microfluidic system capable of aligning, immobilizing, and precisely compressing hundreds of Drosophila melanogaster embryos with minimal user intervention. This system enables high-resolution imaging and recovery of samples for post-stimulation analysis and can be scaled to accommodate other multicellular biological systems.

During embryogenesis, coordinated cell movement generates mechanical forces that regulate gene expression and activity. To study this process, tools such ...

Automated Multimodal Stimulation and Simultaneous Neuronal Recording from Multiple Small Organisms

Fluorescent genetically encoded calcium indicators have contributed greatly to our understanding of neural dynamics from the level of individual neurons to entire brain circuits. However, neural responses may vary due to prior experience, internal states, or stochastic factors, thus generating the need for methods that can assess neural function across many individuals at once. Whereas most recording techniques examine a single animal at a time, we describe the use of wide-field microscopy to scale up neuronal recordings to dozens of Caenorhabditis elegans or other sub-millimeter-scale organisms at once. Open-source hardware and software allow great flexibility in programming fully automated experiments that control the intensity and timing of various stimulus types, including chemical, optical, mechanical, thermal, and electromagnetic stimuli. In particular, microfluidic flow devices provide precise, repeatable, and quantitative control of chemosensory stimuli with sub-second time resolution. The NeuroTracker semi-automated data analysis pipeline then extracts individual and population-wide neural responses to uncover functional changes in neural excitability and dynamics. This paper presents examples of measuring neuronal adaptation, temporal inhibition, and stimulus crosstalk. These techniques increase the precision and repeatability of stimulation, allow the exploration of population variability, and are generalizable to other dynamic fluorescent signals in small biosystems from cells and organoids to whole organisms and plants.

We present a method for the flexible chemical and multimodal stimulation and recording of simultaneous neural activity from many Caenorhabditis elegans worms. This method uses microfluidics, open-source hardware and software, and supervised automated data analysis to enable the measurement of neuronal phenomena such as adaptation, temporal inhibition, and stimulus crosstalk.

Fluorescent genetically encoded calcium indicators have contributed greatly to our understanding of neural dynamics from the level of individual neurons ...

Constructing and Operating a Cooling Stage to Immobilize C. elegans En Masse

Assembly and Operation of a Cooling Stage to Immobilize C. elegans on Their Culture Plates

High-resolution in vivo microscopy approaches can reveal subtle information and fine details inside the model animal Caenorhabditis elegans (C. elegans), but require strong animal immobilization to prevent motion blur in the images. Unfortunately, most current immobilization techniques require substantial manual effort, rendering high-resolution imaging low-throughput. Immobilization of C. elegans is greatly simplified by using a cooling approach that can easily immobilize entire populations directly on their cultivation plates. The cooling stage can establish and maintain a wide range of temperatures with a uniform distribution on the cultivation plate. In this article, the whole process of building the cooling stage is documented. The aim is that a typical researcher can build an operational cooling stage in their laboratory following this protocol without difficulty. Utilization of the cooling stage following three protocols is shown, and each protocol has advantages for different experiments. Also shown is an example cooling profile of the stage as it approaches its final temperature and some helpful tips in using cooling immobilization.

Author Spotlight: Assembly and Operation of a Cooling Stage to Immobilize C. elegans on Their Culture Plates  

This paper describes protocols for constructing and operating a cooling stage to immobilize C. elegans on their original cultivation plates en masse.

High-resolution in vivo microscopy approaches can reveal subtle information and fine details inside the model animal Caenorhabditis elegans (C. elegans), ...

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

Samenvatting

Meer video's verkennen