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

作者感谢 Atsushi Higashitani 博士就线虫和 Yuya hari 博士、Yasuhiko Yokota 和 Kobayashi 康彦的治疗提出的善意建议, 并进行了宝贵的讨论。作者感谢 Caenorhabditis 遗传中心提供了线虫和大肠杆菌菌株。我们感谢 QST-Takasaki 的 TIARA 回旋加速器的机组人员对辐照实验的善意帮助。我们感谢苏珊·弗尼斯博士编辑了这份手稿的草稿。这项研究得到了从 JPS 到 m. s. 的 KAKENHI (赠款号码 JAK15K11921 和 J18K1839) 的部分支持。

1. 应变和维护
<ol><li>根据实验目的, 选择合适的线虫和大肠杆菌(食品) 菌株。 注: 在本文件中, 一般使用野生型 N2-10c.线虫 (图 2a), hbr4:3:goeIs3[pmyo: Gcamp3.35: unc-54-3 ' utr, 无 119(+)]V11仅用于成像分析。大肠杆菌op50 被用作线虫的食物。一些体型异常的突变体, 如卷状的 119(e2498) iii 突?...

<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>

在带电条件下, 使用可湿性 PDMS 微流体芯片对线虫进行片上固定, 使多种动物能够有效地进行有针对性的微束照射。易于操作和防止干燥的特点使该系统不仅适用于微束照射, 而且适用于几种行为检测。这些蠕虫单已经商业化, 可以很容易地获得。传统的微流体芯片, 如嗅觉芯片, 与诸如在封闭的微流体通道中堵塞动物和鸡蛋等问题有关, 使它们难以采集动物, 因此, 这种芯片往往是一次性的, ?...

辐射, 包括 x 射线、伽马射线和重离子束, 广泛用于生物应用, 如癌症诊断和治疗, 以及离子束育种。目前, 许多研究和技术发展的重点是辐射的影响1、2、3。微束照射是识别生物体中放射性敏感位点的有力手段 4.美国量子与放射科学与技术研究所 (QST-Takasaki) 高崎高级辐射研究所 (QST-Takasaki) 一直在开发一种利用重离子在显微镜下照射单个细胞的技术微束5, 并建立了方法, 使目标微束照射的几个模型动物, 如线虫线虫4,6, 蚕7和oryzias纬度(日本美达卡)8。线虫线虫的靶向微束照射可以有效地击倒特定区域, 如头部区域的神经环, 从而有助于确定这些系统在运动等过程中的作用。
开发了一种不需要麻醉的线虫人片上固定化的方法, 以允许微束照射 4.此外, 为了改进上一项研究4中使用的微流体芯片, 我们最近开发了可湿性、离子渗透、聚二甲基硅氧烷 (PDMS) 微流体芯片, 称为蠕虫片 (见材料表), 用于固定线虫人9。这些包括超薄软片 (厚度 = 300μm; 宽度 = 15 毫米; 长度 = 15 毫米), 表面有多个 (20 或 25) 直微流体通道 (深度 = 70μm; 宽度 = 60μm 或 50μm; 长度 = 8 mm) (图 1a-d)。微流体通道是开放的, 允许多个动物同时被封闭在其中 (图 1e)。这些薄片具有弹性, 可以扩大微流体通道 (约 10%,图 1f), 弹性允许动物轻轻包裹。此外, 由于 PDMS 的自吸附能力, 动物可以通过用薄膜覆盖蠕虫片的表面, 在其中不被推入通道进行围护结构, 从而将动物密封在通道中。通过翻拍封面片, 我们可以很容易地收集动物。
这些通道在被封闭或收集时不会伤害虫子。此外, 板材是由 PDMS, 这基本上是疏水, 但水的保持可以通过赋予亲水性的材料。水的保留性和厚度是蜗杆板的良好特性。保水能力可防止动物在长时间固定后脱水, 并可进行长期观察。
此外, 如前所述, 板材只有300μm 厚, 允许重离子, 如碳离子 (水的范围约为1毫米) 通过片状包围动物。这样就可以检测离子粒子, 准确测量所施加的辐射剂量。此外, 蠕虫板可以重复使用, 因此是经济的。用传统的注射方法, 封闭的动物有时会死亡, 不能被带出通道;他们的蛋也会堵塞频道。这使得芯片无法使用。因此, 芯片基本上是一次性的, 成本效益比率很低。
本文详细介绍了用蠕虫片对活体线虫进行片上固定化的一系列方法。通过对片上固定3小时后动物的运动, 对合适的覆盖膜进行了评价。此外, 我们还展示了用于成像观测和微束照射的片上固定化示例。

<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

辐射被广泛用于生物应用和离子束育种, 在这些方法中, 微束辐射是识别活生物体中放射性敏感位点的有力手段。本文介绍了为线虫活体个体的靶向微束照射而开发的一系列片上固定方法。值得注意的是, 我们以前开发的聚二甲基硅氧烷 (PDMS) 微流体芯片的治疗方法是在不需要麻醉的情况下固定线虫个体的。这种芯片, 被称为蠕虫片, 是弹性, 允许微流体通道被扩大, 弹性允许动物被轻轻包围。此外, 由于 PDMS 的自吸附能力, 动物可以通过用薄膜覆盖蠕虫片的表面, 在其中不被推入通道进行围护结构, 从而将动物密封在通道中。通过翻拍封面片, 我们可以很容易地收集动物。此外, 蠕虫片显示水的保留, 并允许线虫人在活的条件下进行长时间的微观观察。此外, 板材厚度仅为 300μm, 允许碳离子等重离子穿过封闭动物的片状物, 从而可检测离子粒子并准确测量所施加的辐射剂量。由于用于围住动物的封面膜的选择对成功的长期固定非常重要, 我们对合适的封面片进行了选择, 并在一些电影中展示了推荐的封面电影。作为芯片的应用实例, 我们介绍了动物包围蜗杆片微流体通道的肌肉活动的成像观测, 以及微束照射。这些例子表明, 蠕虫板极大地扩大了生物实验的可能性。

活性的线虫个体可以使用超薄、可湿 PDMS、微流体芯片 (蜗杆片) 成功固定化。我们研究了不同盖膜密封蜗杆片的适用性, 如协议第3节所述。为了评估覆盖膜的密封效果, 我们分别使用盖板玻璃 (厚度: 130-170μm)、PET 薄膜 (厚度: 125μm) 和 PS 膜 (厚度约 130μm) 确定了片上固定3小时后动物的运动力。如图 5所示, 允许自由移动3小时的控制动物和用 ps 膜...

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

超薄聚二甲基硅氧烷微氟化片在保水作用下固定化活线虫个体

建立了一系列固定化方法, 允许使用最近开发的具有水保留的超薄聚二甲基硅氧烷微流体芯片对活的虫进行定向照射。这种新颖的片上固定也足以进行成像观测。详细说明了该芯片的处理和应用实例。

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

此协议的一个优点是蠕虫板易于处理。该表具有防止干燥的功能，使我们能够对活的动物进行微拉姆照射，进行多种行为分析，并长期观察。此蠕虫片中的微流体通道是开放的，因此更容易收集动物。这些蠕虫片也可以反复使用，使它们更经济。当我们开发这种技术时，我们想用显微镜下微米照射动物的一部分。所以我们需要清晰，非常薄的芯片能够举行动物。首先，在舞台上放置一张薄透明薄的薄板，用作底盖膜。用扁平钳子轻轻将蠕虫片放在底盖膜上。将至少三滴缓冲液放在 6 厘米未处理的培养皿表面。然后，从文化盘子里挑选几个成年的C.elegans，用一个普拉蒂娜采摘机。将动物转移到一个液滴与普拉蒂娜采摘器，并允许动物在缓冲液中游泳，以去除任何细菌。将动物用两个单独的液滴清洗，并在另一个液滴中冲洗动物。接下来，将缓冲液滴放在蠕虫片表面。从缓冲液滴中去除被洗过的动物，并将其转移到虫片上的液滴。使用扁平钳子将 PS 盖膜放在蠕虫片上，然后轻轻地按压从纸片一端到另一端的通道。或者简单地将动物放入水滴中，用盖膜轻轻密封，使水滴在覆盖和蠕虫片之间扩散，使动物在通道内。通过显微镜检查1-2倍放大倍率的运动，确认动物是否活着。记录动物的位置，并特别记录每个动物被封闭的通道数，以及每个动物在通道中的位置。要收集固定动物，使用扁平钳子从虫片中去除覆盖膜，然后将 10-15 微升缓冲液滴到包围动物的微流体通道之一。在立体显微镜下，观察动物开始旋转。然后使用普拉蒂娜拾取器拾起游泳的动物，并把它们转移到一个检测板。在像先前证明的封闭动物之后，用荧光显微镜观察动物身上的荧光点。然后利用视频采集图像钙离子波传播，观察动态活动。将用过的蠕虫片放在六厘米的培养皿上，并在片上放置约 100 微升灭菌的超纯水。使用戴手套的手指将水铺在床单表面，并清除任何污染物。接下来，使用一次性湿巾去除虫片中的水分。将五毫升70%乙醇分配到培养皿中，并使用戴手套的手指将乙醇涂在蠕虫片上。最后，在薄板干燥后，将薄片放在无菌培养皿和盖子上。利用本协议，对不同封面电影的适用性进行了调查。用PS薄膜封闭在微流体通道中的对照动物和动物之间的运动性没有显著差异。相比之下，在封闭在盖玻璃下的动物中，运动性显著降低。用宠物膜封闭的动物的动性也显著下降。仔细选择覆盖膜以密封微流体通道非常重要。使用该协议，我们评估了不同覆盖膜的适用性，通过运动测定动物三个小时后，片上固定。利用这项技术，我们打算开发一种超薄的可湿芯片，仅用于微米辐照。然而，这些蠕虫表已经用于成像和行为检测，超出其原始应用程序。

Research

JoVE Journal

Bioengineering

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

Fluorescence detection methods for microfluidic droplet platforms

荧光检测方法，为微流体液滴平台

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

科研

生物工程

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

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

基于微流体Electrotaxis的，为按需定量分析<em&gt;线虫</em&gt;&#39;LOCOMOTION的

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

微流控芯片的制备及方法检测甲型流感

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

一个微流控芯片单细胞的多功能化学分析

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

微流体芯片ICPMS进样

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

使用多通道双电场微流控芯片肺癌细胞Electrotaxis研究

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

超滤/纳滤膜性能测试三维印刷微流控横流系统

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

自动化脂双层膜的形成使用聚二甲基硅氧烷薄膜

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

活细胞层的快速消减带图案微流控探头

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

细胞迁移研究在垂直化学和氧梯度聚二甲基硅氧烷的聚碳酸酯微流体装置

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