Name: immobilization of live caenorhabditis elegans individuals using an ultra-thin polydimethylsiloxane microfluidic chip with water retention
Uploaded: 2019-03-19T14:00:00
Description: Watch this Scientific Journal Video about Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention at JoVE.com

The authors thank Dr. Atsushi Higashitani for kind advice regarding treatment of C. elegans and Drs. Yuya Hattori, Yuichiro Yokota, and Yasuhiko Kobayashi for valuable discussions. The authors thank the Caenorhabditis Genetic Center for providing strains of C. elegans and E. coli. We thank the crew of the cyclotron of TIARA at QST-Takasaki for their kind assistance with the irradiation experiments. We thank Dr. Susan Furness for editing a draft of this manuscript. This study was supported in part by KAKENHI (Grant Numbers JP15K11921 and JP18K18839) from JSPS to M.S.

1. Strains and maintenance
<ol>
	<li>Select a suitable strain of C. elegans and Escherichia coli (food) depending on the purpose of the experiment. 
	NOTE: In the present paper, wild-type N210C. elegans (Figure 2A) is generally used, and HBR4:goeIs3[pmyo-3::GCamP3.35::unc-54 - 3&#39;utr, unc-119(+)]V11 is only employed for imaging assay. E. coli OP50 was used as food for C...

<ol>
	<li>Funayama, T., Hamada, N., Sakashita, T., Kobayashi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heavy-Ion+microbeams-development+and+applications+in+biological+studies.">Heavy-Ion microbeams-development and applications in biological studies.</a> IEEE Transactions on Plasma Science. 36 (4), 1432-1440 (2008).</li><li>Tanaka, A., Shikazono, N., Hase, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+biological+effects+of+ion+beams+on+lethality,+molecular+nature+of+mutation,+mutation+rate,+and+spectrum+of+mutation+phenotype+for+mutation+breeding+in+higher+plants.">Studies on biological effects of ion beams on lethality, molecular nature of mutation, mutation rate, and spectrum of mutation phenotype for mutation breeding in higher plants.</a> Journal of Radiation Research. 51 (3), 223-233 (2010).</li><li>Ghita, M., Fernandez-Palomo, C., Fukunaga, H., Fredericia, P. M., Schettino, G., Br&#228;uer-Krisch, E., Butterworth, K. T., McMahon, S. J., Prise, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbeam+evolution:+from+single+cell+irradiation+to+pre-clinical+studies.">Microbeam evolution: from single cell irradiation to pre-clinical studies.</a> International Journal of Radiation Biology. 94 (8), 708-718 (2018).</li><li>Suzuki, M., Hattori, Y., Sakashita, T., Yokota, Y., Kobayashi, Y., Funayama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Region-specific+irradiation+system+with+heavy-ion+microbeam+for+active+individuals+of+Caenorhabditis+elegans.">Region-specific irradiation system with heavy-ion microbeam for active individuals of Caenorhabditis elegans.</a> Journal of Radiation Research. 58 (6), 881-886 (2017).</li><li>Funayama, T., Wada, S., Yokota, Y., Fukamoto, K., Sakashita, T., Taguchi, M., Kakizaki, T., Hamada, N., Suzuki, M., Furusawa, Y., Watanabe, H., Kiguchi, K., Kobayashi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heavy-ion+microbeam+system+at+JAEA-Takasaki+for+microbeam+biology.">Heavy-ion microbeam system at JAEA-Takasaki for microbeam biology.</a> Journal of Radiation Research. 49 (1), 71-82 (2008).</li><li>Sugimoto, T., Dazai, K., Sakashita, T., Funayama, T., Wada, S., Hamada, N., Kakizaki, T., Kobayashi, Y., Higashitani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cycle+arrest+and+apoptosis+in+Caenorhabditis+elegans+germline+cells+following+heavy-ion+microbeam+irradiation.">Cell cycle arrest and apoptosis in Caenorhabditis elegans germline cells following heavy-ion microbeam irradiation.</a> International Journal of Radiation Biology. 82 (1), 31-38 (2006).</li><li>Fukamoto, K., Shirai, K., Sakata, T., Sakashita, T., Funayama, T., Hamada, N., Wada, S., Kakizaki, T., Shimura, S., Kobayashi, Y., Kiguchi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+irradiation+method+for+the+first+instar+silkworm+larvae+using+locally+targeted+heavy-ion+microbeam.">Development of the irradiation method for the first instar silkworm larvae using locally targeted heavy-ion microbeam.</a> Journal of Radiation Research. 48 (3), 247-253 (2007).</li><li>Yasuda, T., Kamahori, M., Nagata, K., Watanabe-Asaka, T., Suzuki, M., Funayama, T., Mitani, H., Oda, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abscopal+activation+of+microglia+in+embryonic+fish+brain+following+targeted+irradiation+with+heavy-ion+microbeam.">Abscopal activation of microglia in embryonic fish brain following targeted irradiation with heavy-ion microbeam.</a> International Journal of Molecular Sciences. 18 (7), 1-15 (2017).</li><li>Suzuki, M., Sakashita, T., Hattori, Y., Yokota, Y., Kobayashi, Y., Funayama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+ultra-thin+chips+for+immobilization+of+Caenorhabditis+elegans+in+microfluidic+channels+during+irradiation+and+selection+of+buffer+solution+to+prevent+dehydration.">Development of ultra-thin chips for immobilization of Caenorhabditis elegans in microfluidic channels during irradiation and selection of buffer solution to prevent dehydration.</a> Journal of Neuroscience Methods. 306, 32-37 (2018).</li><li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+Caenorhabditis+elegans.">The genetics of Caenorhabditis elegans.</a> Genetics. 77 (1), 71-94 (1974).</li><li>Schwarz, J., Spies, J. P., Bringmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+muscle+contraction+and+a+relaxed+posture+during+sleep-like+Lethargus.">Reduced muscle contraction and a relaxed posture during sleep-like Lethargus.</a> Worm. 1 (1), 12-14 (2012).</li><li>Saeki, S., Yamamoto, M., Iino, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+of+chemotaxis+revealed+by+paired+presentation+of+a+chemoattractant+and+starvation+in+the+nematode+Caenorhabditis+elegans.">Plasticity of chemotaxis revealed by paired presentation of a chemoattractant and starvation in the nematode Caenorhabditis elegans.</a> Experimental Biology. 204, 1757-1764 (2001).</li><li>Sawin, E. R., Ranganathan, R., Horvitz, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C.+elegans+locomotory+rate+is+modulated+by+the+environment+through+a+dopaminergic+pathway+and+by+experience+through+a+serotonergic+pathway.">C. elegans locomotory rate is modulated by the environment through a dopaminergic pathway and by experience through a serotonergic pathway.</a> Neuron. 26 (3), 619-631 (2000).</li><li>Momma, K., Homma, T., Isaka, R., Sudevan, S., Higashitan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat-induced+calcium+leakage+causes+mitochondrial+damage+in+Caenorhabditis+elegans+body-wall+muscles.">Heat-induced calcium leakage causes mitochondrial damage in Caenorhabditis elegans body-wall muscles.</a> Genetics. 206 (4), 1985-1994 (2017).</li><li>Kerr, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+activity+of+neurons+and+muscles.">Imaging the activity of neurons and muscles.</a> WormBook. 2, 1-13 (2006).</li><li>Aubry, G., Lu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+perspective+on+optical+developments+in+microfluidic+platforms+for+Caenorhabditis+elegans+research.">A perspective on optical developments in microfluidic platforms for Caenorhabditis elegans research.</a> Biomicrofluidics. 8, 011301 (2014).</li><li>Lumirror Catalog. TORAY Available from: <a target="_blank" href="https://www.toray.jp/films/en/products/pdf/lumirror.pdf">https://www.toray.jp/films/en/products/pdf/lumirror.pdf</a> (2018)</li><li>Otobe, K., Itou, K., Mizukubo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-moulded+substrates+for+the+analysis+of+structure-dependent+behaviour+of+nematodes.">Micro-moulded substrates for the analysis of structure-dependent behaviour of nematodes.</a> Nematology. 6 (1), 73-77 (2004).</li><li>Chronis, N., Zimmer, M., Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics+for+in+vivo+imaging+of+neuronal+and+behavioral+activity+in+Caenorhabditis+elegans.">Microfluidics for in vivo imaging of neuronal and behavioral activity in Caenorhabditis elegans.</a> Nature Methods. 4 (9), 727-731 (2007).</li><li>Hulme, S. E., Shevkoplyas, S. S., Apfeld, J., Fontana, W., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microfabricated+array+of+clamps+for+immobilizing+and+imaging+C.+elegans.">A microfabricated array of clamps for immobilizing and imaging C. elegans.</a> Lab on a Chip. 7 (11), 1515-1523 (2007).</li><li>Lockery, S. R., Lawton, K. J., Doll, J. C., Faumont, S., Coulthard, S. M., Thiele, T. R., Chronis, N., McCormick, K. E., Goodman, M. B., Pruitt, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+dirt:+Microfluidic+substrates+for+nematode+neurobiology+and+behavior.">Artificial dirt: Microfluidic substrates for nematode neurobiology and behavior.</a> Journal of Neurophysiology. 99 (6), 3136-3143 (2008).</li><li>Gilleland, C. L., Rohde, C. B., Zeng, F., Yanik, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+immobilization+of+physiologically+active+Caenorhabditis+elegans.">Microfluidic immobilization of physiologically active Caenorhabditis elegans.</a> Nature Protocols. 5 (12), 1888-1902 (2010).</li><li>Fehlauer, H., Nekimken, A. L., Kim, A. A., Pruitt, B. L., Goodman, M. B., Krieg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+a+microfluidics+device+for+mechanical+stimulation+and+high+resolution+imaging+of+C.+elegans.">Using a microfluidics device for mechanical stimulation and high resolution imaging of C. elegans.</a> Journal of Visualized Experiments. (132), e56530 (2018).</li></ol>

On-chip immobilization of C. elegans under live conditions using a wettable PDMS microfluidic chip enables the efficient targeted microbeam irradiation of multiple animals. The ease of handling and features to prevent drying make this system suitable for applications not only in microbeam irradiation, but also in several behavioral assays. These worm sheets have already been commercialized and can be easily obtained. Conventional microfluidic chips, such as olfactory chips, are associated with problems including...

Radiation, including X-rays, gamma rays, and heavy-ion beam, is widely used for biological applications such as in cancer diagnosis and treatment, and for ion-beam breeding. Numerous studies and technical developments are currently focusing on the effects of radiation1,2,3. Microbeam irradiation is a powerful means of identifying radiosensitive sites in living organisms4. The Takasaki Advanced Radiation Research Institute of National Institutes for Quantum and Radiological Science and Technology (QST-Takasaki) has been developing a technology to irradiate individual cells under microscopic observation using heavy-ion microbeams5, and has established methods to enable targeted microbeam irradiation of several model animals, such as the nematode Caenorhabditis elegans4,6, silkworms7, and Oryzias latipes (Japanese medaka)8. Targeted microbeam irradiation of the nematode C. elegans allows the effective knockdown of specific regions, such as the nerve ring in the head region, thus helping to identify the roles of these systems in processes such as locomotion.
A method for on-chip immobilization of C. elegans individuals without the need for anesthesia has been developed to allow for microbeam irradiation4. In addition, to improve microfluidic chips used in the previous study4, we have recently developed wettable, ion-penetrable, polydimethylsiloxane (PDMS) microfluidic chips, referred to as worm sheets (see Table of Materials), for immobilizing C. elegans individuals9. These comprise of ultra-thin soft sheets (thickness = 300 &#181;m; width = 15 mm; length = 15 mm) with multiple (20 or 25) straight microfluidic channels (depth = 70 &#181;m; width = 60 &#181;m or 50 &#181;m; length = 8 mm) at the surface (Figure 1A-D). The microfluidic channels are open and allow multiple animals to be enclosed in them simultaneously (Figure 1E). The sheets are resilient to allow the microfluidic channels to be expanded (by ~10%, Figure 1F), 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.
The channels do not hurt the worms when they are being enclosed or when they are collected. Furthermore, the sheets are made from PDMS, which is essentially hydrophobic, but water retention can be achieved by imparting hydrophilicity to the material. The water retention and thickness are favorable characteristics of the worm sheets. The water-retention capacity prevents dehydration of the animals after prolonged immobilization and enables long-term observations to be carried out.
In addition, as described previously9, the sheets are only 300 &#181;m thick, allowing heavy ions such as carbon ions (with a range of about 1 mm in water) to pass through the sheet enclosing the animals. This allows the ion particles to be detected and the applied radiation dose to be measured accurately. Moreover, the worm sheets can be reused and are thus economical. With the conventional injection method, the animals enclosed are sometimes dead and they cannot be taken out of the channel; their eggs can also clog the channels. This makes the chip unusable. Chips are, therefore, basically disposable and the cost-benefit ratio is poor.
In the present paper, we describe in detail a series of methods for on-chip immobilization of live C. elegans individuals using worm sheets. Through locomotion assays of animals 3 h after on-chip immobilization, we evaluated the suitable cover film. In addition, we showed the examples of on-chip immobilization for both imaging observations and microbeam irradiation.

<table border="0" cellpadding="0" cellspacing="0" style="width:1151px;" width="1152">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">C. elegans wild-type strain</td>
			<td style="width:125px;">Caenorhabditis Genetics Center (CGC) , Minnesota, USA</td>
			<td style="width:353px;">N2</td>
			<td style="width:449px;">Wild-type C. elegans strain generally used in this study</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">C. elegans unc-119(e2498) III mutant strain</td>
			<td style="width:125px;">Caenorhabditis Genetics Center (CGC) , Minnesota, USA</td>
			<td style="width:353px;">CB4845</td>
			<td style="width:449px;">C. elegans strain only employed as an example of mutants with abnormal body shape&#160;</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:223px;">C. elegans transgenic strain HBR4</td>
			<td style="width:125px;">Caenorhabditis Genetics Center (CGC) , Minnesota, USA</td>
			<td style="width:353px;">HBR4</td>
			<td style="width:449px;">The genotype of this transgenic C. elegans strain is HBR4:goeIs3[pmyo-3::GCamP3.35:: unc-54&#8211;3&#8217;utr, unc-119(+)]V. This strain was only employed for imaging observation.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">E. coli strain</td>
			<td style="width:125px;">Caenorhabditis Genetics Center (CGC) , Minnesota, USA</td>
			<td style="width:353px;">OP50</td>
			<td style="width:449px;">E. coli strain used as food for C. elegans</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Worm Sheet IR (50/60)</td>
			<td style="width:125px;">Biocosm, Inc., Hyogo, Japan</td>
			<td>BCM17-0001</td>
			<td style="width:449px;">Microfluidic chip with 25 straight 50/60-&#181;m width channels used in all experiments and observation in this paper&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Worm Sheet 60</td>
			<td style="width:125px;">Biocosm, Inc., Hyogo, Japan</td>
			<td>BCM18-0001</td>
			<td style="width:449px;">Microfluidic chip with 20 straight 60 &#181;m-width channels. This is sitable for adults 3-5 days after hatching at 20&#176;C.&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Worm Sheet 50</td>
			<td style="width:125px;">Biocosm, Inc., Hyogo, Japan</td>
			<td>BCM18-0002</td>
			<td style="width:449px;">Microfluidic chip with 20 straight 50 &#181;m-width channels. This is sitable for youg adults ~3 days after hatching at 20&#176;C.&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">MICRO COVER GLASS</td>
			<td style="width:125px;">MATSUNAMI GLASS IND. LTD.</td>
			<td>C030401</td>
			<td style="width:449px;">Cover glass (thickness: 130-170 &#181;m) used in locomotion assays in Protocol 3</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Polystyrene Film</td>
			<td style="width:125px;">Biocosm, Inc., Hyogo, Japan</td>
			<td>BCM18-0001/ BCM18-0002</td>
			<td style="width:449px;">Bundled items of Worm Sheets. PS filim (thickness: ~130 &#181;m) used in locomotion assays in Protocol 3.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Polyester Film Lumirror</td>
			<td style="width:125px;">TORAY INDUSTRIES, INC., Tokyo, Japan</td>
			<td>Lumirror T60 (t 125 &#181;m)</td>
			<td style="width:449px;">PET filim (thickness: 125 &#181;m) used in locomotion assays in Protocol 3</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">IWAKI 60 mm/non-treated dish</td>
			<td style="width:125px;">AGC Techno Glass Co., Ltd., Shizuoka, Japan).</td>
			<td>1010-060</td>
			<td style="width:449px;">Non-treated dish used in incuvation of C. elegans in Protocol 1</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">IWAKI 35 mm/non-treated dish</td>
			<td style="width:125px;">AGC Techno Glass Co., Ltd., Shizuoka, Japan).</td>
			<td>1010-035</td>
			<td style="width:449px;">Non-treated dish used in locomotion assays in Protocol 3</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Milli-Q</td>
			<td style="width:125px;">Merck, France</td>
			<td></td>
			<td style="width:449px;">Ultrapure water</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Kimwipe S-200</td>
			<td style="width:125px;">Nippon Paper Crecia Co., Ltd., Tokyo, Japan</td>
			<td>62020</td>
			<td style="width:449px;">120 mm x 215 mm; 200 sheets/ box</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">WormStuff Worm Pick</td>
			<td style="width:125px;">Genesee Scientific Corporation, CA, USA)</td>
			<td>59-AWP</td>
			<td style="width:449px;">Platina picker specilized for picking up C. elegans</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Research Stereo Microscope System</td>
			<td style="width:125px;">OLYMPUS CORPORATION, Tokyo, Japan</td>
			<td>SZX16</td>
			<td style="width:449px;">Micriscope used in all experiments and observation in this paper</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Motorized Focus Stand for SZX16</td>
			<td style="width:125px;">OLYMPUS CORPORATION, Tokyo, Japan</td>
			<td style="width:353px;">SZX2-ILLB</td>
			<td style="width:449px;">This was used for bright field observation in Protocol 3-8.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Objective Lens (&#215;1)</td>
			<td style="width:125px;">OLYMPUS CORPORATION, Tokyo, Japan</td>
			<td>SDFPLAPO1&#215;PF</td>
			<td style="width:449px;">NA: 0.15; W.D.: 60 mm. This lends was used for bright field observation in Protocol 3-8.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Objective Lens (&#215;2)</td>
			<td style="width:125px;">OLYMPUS CORPORATION, Tokyo, Japan</td>
			<td>SDFPLAPO2XPFC</td>
			<td style="width:449px;">NA: 0.3; W.D.: 20 mm. This&#160; lends was used for imaging observations.</td>
		</tr>
	</tbody>
</table>

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.

Active C. elegans individuals could be immobilized successfully using an ultra-thin, wettable PDMS, microfluidic chip (worm sheet). We investigated the suitability of different cover films for sealing the worm sheet, as described in protocol section 3. To evaluate the sealing effects of the cover films, we determined the motility of animals 3 h after on-chip immobilization using cover glass (thickness: 130-170 &#181;m), PET film (thickness: 125 &#181;m), and PS film (thickness: ~...

Watch this Scientific Journal Video about Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention at JoVE.com

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.

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 ...

Research

JoVE Journal

Bioengineering

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

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

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 ...

Microfluidic Chip Fabrication and Method to Detect Influenza

Fast  and effective diagnostics play an important role in controlling infectious  disease by enabling effective patient management and treatment. Here, we ...

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

A Microfluidic Chip for ICPMS Sample Introduction

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma ...

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

Three-Dimensionally Printed Microfluidic Cross-flow System for Ultrafiltration/Nanofiltration Membrane Performance Testing

Minimization and management of membrane fouling is a formidable challenge in diverse industrial processes and other practices that utilize membrane ...

Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for ...

Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one ...

Polydimethylsiloxane-polycarbonate Microfluidic Devices for Cell Migration Studies Under Perpendicular Chemical and Oxygen Gradients

This paper reports a microfluidic device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film to study cell migration under ...