这项研究得到了国家科学基金会的支持, 通过赠款 PHY-1205494 和 MCB-1413134 (EL) 和韩国国家研究基金会 2017R1D1A1B03035671 (库特勒)。

下面的协议使用 WormSpa5, 以前描述的微流控器用于对蠕虫进行纵向成像。WormSpa 的制作 (从 CAD 文件开始, 可以根据要求从作者获得) 是简单的, 但需要一些专门知识。在大多数情况下, 制造可以很容易地由核心设施或提供此类服务的商业公司完成。在制作设备时, 请确保指定特征的高度为50µm。
1. 实验设置
<ol><li>将微流控装置安装在具有机动化阶段的倒置荧光显微镜上。</li>
	<li>将一个轨道振动筛靠近显微镜, 但要确保振动筛的振动不会直接影响显微镜。例如, 将振动筛放在固定在墙上的搁板上, 使其与光学表没有接触。</li>
	<li>将注射器泵放在轨道振动筛上。将泵带到振动筛上, 使其在晃动时不摇晃。</li>
</ol>2. 微流控环境的制备
注: 在实验中, 四注射器通过注射器管连接到微流控装置, 如图 1所述。这一步描述了这些注射器的制备。除非另有提及, 保持注射器管尽可能短, 而不使其绷紧。
<ol><li>准备一个出口注射器和一个注射器管。此注射器 (图 1中的 S1) 稍后将通过注射器管连接到设备的出口, 并用于临时存储从设备中流出的液体。<ol>
<li>通过去除20克 x 1/2 英寸注射器尖端的塑料部件来制作适配器针, 只保留针 (金属部分)。这样做, 通过持有的塑料和金属的部分钳子和拉他们分开。针上残留的塑料残渣可以用本生燃烧器烧掉。</li>
		<li>根据实验装置的几何形状弯曲针头, 同时用钳子握住两端。在这里描述的设计中, 弯曲的针在它的中间到一个角度的〜110°是最方便的。</li>
		<li>将适配器针插入到管子的一半。另一半稍后将插入设备。注射器管应与20克无泄漏的注射器尖端兼容 (例如, 油管内径为0.86 毫米, 外径为1.32 毫米)。推荐的管长为50厘米。</li>
		<li>通过20克 x 1/2 英寸的注射器尖, 将10毫升鲁尔锁注射器连接到注射器管的另一端 (打开) 端。</li>
	</ol>
</li>
	<li>准备缓冲入口注射器和注射器管。其中一个注射器 (在图 1中的 S2) 被用作进入设备并控制注入流的流体的蓄水池。另一个注射器 (图 1中的 S3) 是缓冲区交换所必需的, 如步骤3.2 所述。<ol>
<li>将10毫升鲁尔锁注射器 (S2) 直接连接到3通阀, 并通过注射器管将另一注射器 (S3) 连接到同一阀门上 (图 1): 通过注射器尖端连接 S3 到管, 并通过另一个注射器提示连接到阀门的管。 注: 这些材料的连接顺序无关紧要。</li>
		<li>将注射器管连接到3路阀门的剩余端口。在步骤2.1.2 中准备另一个适配器针, 并将针插入到注射器管的另一端 (打开) 端。</li>
		<li>设置阀门, 使步骤2.2.2 和 S2 中的注射器管在 S3 关闭时连接 (图 1)。</li>
	</ol>
</li>
	<li>准备一个蠕虫入口注射器和一个注射器管。此注射器 (图 1中的 S4) 用于将蠕虫加载到设备中。<ol>
<li>连接一个长的注射器管到一个10毫升鲁尔锁注射器 (S4)。从将用于装货的液体体积中确定该管的长度;例如, 100 厘米的油管支持超过2毫升的液体 (也见步骤 4.2)。</li>
		<li>在步骤2.1.2 中准备另一个适配器针, 并将一针的一半插入注射器管的另一端 (打开) 端。</li>
	</ol>
</li>
	<li>用 s-介质12冲洗所有注射器和油管两次。用 s-介质填充注射器和管子, 注意去除所有气泡。</li>
	<li>将插座注射器管连接至设备插座。<ol>
<li>将注射器管末端的适配器针插入插座端口。</li>
		<li>通过插座注入少量的 s-介质, 以填满设备, 直到从缓冲入口和蠕虫入口中取出一点 s-介质。</li>
	</ol>
</li>
	<li>将缓冲入口注射器管连接到设备的缓冲入口端口, 同时用一个销 (不锈钢定位销, 直径为1/32 英寸) 堵塞蜗杆入口。</li>
	<li>向设备中注入恒定的缓冲区流。将缓冲入口注射器 S2 到注射器泵上13, 并设置泵, 使 S2 中的介质连续注入到设备中, 流速为3µL/分钟。</li>
</ol>3. 将细菌装入微流控装置
<ol><li>准备大肠杆菌 OP50 悬浮在 s-中等的 12中。<ol>
<li>按照标准的协议, 接种一个250毫升瓶与50毫升磅和一个单一的殖民地, 并孵化过夜37摄氏度。</li>
		<li>离心机的隔夜文化在 4000 rpm 10 分钟, 并并用重悬颗粒在30毫升的 s-培养基。</li>
		<li>通过5µm 注射器过滤器过滤悬浮液。以 600 nm (od600) 的光学密度测量细菌浓度, 并将浓度调整为 OD600 3。这种高密度是为了保持蠕虫的良好喂养。</li>
	</ol>
</li>
	<li>将设备中的缓冲区与 OP50 暂停交换。<ol>
<li>准备一个干净的注射器 (S5), 填充 OP50 悬浮液。垂直地按住注射器, 多次敲击, 收集顶部的气泡。</li>
		<li>打开缓冲入口注射器的3路阀, 关闭与设备的连接, 并连接两个注射器、S2 和 S3。从注射器泵中脱离 S2。</li>
		<li>从阀门上拔下 S2 并将 S5 连接至阀门。把所有在 S5 顶部收集的空气推到 S3 通过阀门, 直到 OP50 的缓冲区进入 S3。在这样做的时候, 确保没有气泡留在 S5 或阀门。</li>
		<li>将3路阀门恢复到原来的位置, 以关闭 S3 和 S5 之间的连接, 并将 S5 连接到设备。将 S5 加载到注射器泵上。打开支撑泵的轨道振动筛。将震动速度设置为大约 200 rpm。</li>
		<li>将流量设置为100µL/分钟。新缓冲区到达设备中的蠕虫所需的时间取决于缓冲入口注射器管的长度 (大约5分钟, 上面描述的油管)。如果需要更快的缓冲交换, 则可以使用更高的流量。</li>
		<li>一旦 OP50 缓冲区在整个设备中传播, 将流速率设置回3µL/分钟。</li>
	</ol>
</li>
</ol>4. 将蠕虫装入微流控装置
<ol><li>准备一个小的低结合离心管 (650 µL), 并填补它与100µL OP50 悬浮。转移大约 20-30 年龄同步的年轻成人蠕虫 (46 小时后 L1 幼虫拘捕在25°c) 从 NGM (线虫生长培养基) 琼脂板到管通过采摘他们一个与蠕虫采摘。可以在标准协议12之后执行年龄同步。</li>
	<li>用 OP50 悬浮液填充蠕虫入口注射器和注射器管 (在图 1中的 S4)。将500µL 的液体注入离心管中。用蠕虫将悬浮液抽出到蠕虫入口注射器管中。请确保注射器管足够长 (> 1 米), 并使被拉的蠕虫留在注射器管, 并没有使所有的方式对注射器 S4。</li>
	<li>将 S4 连接到蠕虫入口。将蠕虫入口注射器管中的所有蠕虫注入微流控装置。拔下 S4 和注射器管。用大头针将蠕虫入口堵塞。</li>
	<li>从机械泵 (图 1) 中松开缓冲入口注射器 S2, 并手动控制 S1 和 S2, 将所有蠕虫推入不同的通道。按下 S2 将蠕虫移动到通道中, 而按下 S1 将移动它们的反向方向。一旦加载了通道, 每个通道中的蠕虫就会阻止其他蠕虫的进入。</li>
	<li>让蠕虫适应新的环境 2-3 小时。</li>
</ol>5. 建立成像协议
<ol><li>查找安全位于设备中的所有蠕虫, 并在控制您的显微镜的图像采集软件中为它们各自设置一个成像位置。请注意, 在某些情况下 (例如当需要更高的放大倍数时, 或者在使用较小的 CCD 传感器的摄像机时), 每个通道都需要多个成像位置。 注意: 虽然可以手动完成此操作, 但某些购置软件包可以加载一个文本文件, 其中列出了应该拍摄图像的位置。由于这些位置是在 WormSpa 中定期订购的, 因此用户可以编写一个小的脚本 (例如Python 或商业软件中的) 来自动创建这样的列表。此脚本的精确格式取决于购置软件所需的文件格式 (请参见辅助材料 1中的示例)。</li>
	<li>设置所需的延时成像频率。例如, 对感染的免疫基因反应的成像是以每帧10分钟的频率进行的。确保所有必要的通道 (例如明亮字段和 GFP 荧光) 都在每个时间点获得。</li>
</ol>6. 寄主对铜绿假单胞菌感染的反应
注意: 这一步是专门研究寄主-病原体相互作用的。或者, 你可以准备一个缓冲区, 其中包含其他感兴趣的环境提示 (生物和非物质的压力源, 药物, 信号分子,等等)。
<ol><li>准备铜绿假单胞菌 PA14 悬浮在 SK 中14中。<ol>
<li>接种一个250毫升的烧瓶, 50 毫升磅和一个单一的殖民地, 并孵化过夜37摄氏度。</li>
		<li>接种另一250毫升的烧瓶, 50 毫升磅和整除的文化, 并在37摄氏度一夜之间孵化。</li>
		<li>离心机的隔夜文化在 4000 rpm 10 分钟, 并并用重悬颗粒在10毫升的 SK 培养基。测量细菌浓度并将浓度调整为 OD600 = 4。</li>
		<li>将悬浮液转移到干净的瓶子上, 在37摄氏度时孵育24小时, 在25摄氏度时晃动24小时。这一步模仿标准杀戮化验中的板块孵化步骤14。</li>
		<li>通过5µm 注射器过滤器过滤悬浮液。这是为了防止细菌聚集堵塞设备。</li>
	</ol>
</li>
	<li>按照与步骤3.2 相同的步骤, 将设备中的 OP50 悬浮液替换为 PA14 悬架。保持成像10小时。</li>
</ol>

<ol>
	<li>Lopez-Maury, L., Marguerat, S., Bahler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuning+gene+expression+to+changing+environments:+from+rapid+responses+to+evolutionary+adaptation.">Tuning gene expression to changing environments: from rapid responses to evolutionary adaptation.</a> Nat Rev Genet. 9 (8), 583-593 (2008).</li><li>de Nadal, E., Ammerer, G., Posas, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+gene+expression+in+response+to+stress.">Controlling gene expression in response to stress.</a> Nat Rev Genet. 12 (12), 833-845 (2011).</li><li>Hulme, S. E., Shevkoplyas, S. S., Samuel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics:+Streamlining+discovery+in+worm+biology.">Microfluidics: Streamlining discovery in worm biology.</a> Nat Methods. 5 (7), 589-590 (2008).</li><li>San-Miguel, A., 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=Microfluidics+as+a+tool+for+C.+elegans+research.">Microfluidics as a tool for C. elegans research.</a> WormBook. , (2013).</li><li>Kopito, R. B., Levine, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Durable+spatiotemporal+surveillance+of+Caenorhabditis+elegans+response+to+environmental+cues.">Durable spatiotemporal surveillance of Caenorhabditis elegans response to environmental cues.</a> Lab Chip. 14 (4), 764-770 (2014).</li><li>Mishra, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+microfluidics+chips+for+live+imaging+and+study+of+injury+Responses+in+Drosophila+larvae.">Using microfluidics chips for live imaging and study of injury Responses in Drosophila larvae.</a> J Vis Exp. (84), e50998 (2014).</li><li>Grisi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMR+spectroscopy+of+single+sub-nL+ova+with+inductive+ultra-compact+single-chip+probes.">NMR spectroscopy of single sub-nL ova with inductive ultra-compact single-chip probes.</a> Sci Rep. 7, 44670 (2017).</li><li>Crane, M. M., Chung, K., Stirman, J., 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=Microfluidics-enabled+phenotyping,+imaging,+and+screening+of+multicellular+organisms.">Microfluidics-enabled phenotyping, imaging, and screening of multicellular organisms.</a> Lab Chip. 10 (12), 1509-1517 (2010).</li><li>Leifer, A. M., Fang-Yen, C., Gershow, M., Alkema, M. J., Samuel, A. D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+manipulation+of+neural+activity+in+freely+moving+Caenorhabditis+elegans.">Optogenetic manipulation of neural activity in freely moving Caenorhabditis elegans.</a> Nat Meth. 8 (2), 147-152 (2011).</li><li>Lee, K. S., Lee, L. E., Levine, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HandKAchip+-+Hands-free+killing+assay+on+a+chip.">HandKAchip - Hands-free killing assay on a chip.</a> Sci Rep. 6, 35862 (2016).</li><li>Lee, K. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonin-dependent+kinetics+of+feeding+bursts+underlie+a+graded+response+to+food+availability+in+C.+elegans.">Serotonin-dependent kinetics of feeding bursts underlie a graded response to food availability in C. elegans.</a> Nat Commun. 8, 14221 (2017).</li><li>Stiernagle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+C.+elegans.">Maintenance of C. elegans.</a> WormBook. , (2006).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> NE-1000 Series of Programmable Syringe Pumps. , (2017).</li><li>Tan, M. W., Mahajan-Miklos, S., Ausubel, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Killing+of+Caenorhabditis+elegans+by+Pseudomonas+aeruginosa+used+to+model+mammalian+bacterial+pathogenesis.">Killing of Caenorhabditis elegans by Pseudomonas aeruginosa used to model mammalian bacterial pathogenesis.</a> Proc Natl Acad Sci U S A. 96 (2), 715-720 (1999).</li><li>Kim, D. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conserved+p38+MAP+kinase+pathway+in+Caenorhabditis+elegans+innate+immunity.">A conserved p38 MAP kinase pathway in Caenorhabditis elegans innate immunity.</a> Science. 297 (5581), 623-626 (2002).</li><li>Troemel, E. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p38+MAPK+regulates+expression+of+immune+response+genes+and+contributes+to+longevity+in+C.+elegans.">p38 MAPK regulates expression of immune response genes and contributes to longevity in C. elegans.</a> PLoS Genet. 2 (11), 183 (2006).</li><li>Estes, K. A., Dunbar, T. L., Powell, J. R., Ausubel, F. M., Troemel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=bZIP+transcription+factor+zip-2+mediates+an+early+response+to+Pseudomonas+aeruginosa+infection+in+Caenorhabditis+elegans.">bZIP transcription factor zip-2 mediates an early response to Pseudomonas aeruginosa infection in Caenorhabditis elegans.</a> Proc Natl Acad Sci U S A. 107 (5), 2153-2158 (2010).</li><li>Byerly, L., Cassada, R. C., Russell, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+life+cycle+of+the+nematode+Caenorhabditis+elegans:+I.+Wild-type+growth+and+reproduction.">The life cycle of the nematode Caenorhabditis elegans: I. Wild-type growth and reproduction.</a> Dev Biol. 51 (1), 23-33 (1976).</li><li>Chalancon, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interplay+between+gene+expression+noise+and+regulatory+network+architecture.">Interplay between gene expression noise and regulatory network architecture.</a> Trends Genet. 28 (5), 221-232 (2012).</li><li>Sanchez, A., Choubey, S., Kondev, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+noise+in+gene+expression.">Regulation of noise in gene expression.</a> Annu Rev Biophys. 42, 469-491 (2013).</li><li>Norman, T. M., Lord, N. D., Paulsson, J., Losick, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochastic+Switching+of+Cell+Fate+in+Microbes.">Stochastic Switching of Cell Fate in Microbes.</a> Annu Rev Microbiol. 69, 381-403 (2015).</li><li>Gardner, T. S., di Bernardo, D., Lorenz, D., Collins, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inferring+genetic+networks+and+identifying+compound+mode+of+action+via+expression+profiling.">Inferring genetic networks and identifying compound mode of action via expression profiling.</a> Science. 301 (5629), 102-105 (2003).</li><li>Samuelson, A. V., Carr, C. E., Ruvkun, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+activities+that+mediate+increased+life+span+of+C.+elegans+insulin-like+signaling+mutants.">Gene activities that mediate increased life span of C. elegans insulin-like signaling mutants.</a> Genes Dev. 21 (22), 2976-2994 (2007).</li><li>Edwards, C. B., Copes, N., Brito, A. G., Canfield, J., Bradshaw, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malate+and+Fumarate+Extend+Lifespan+in+Caenorhabditis+elegans.">Malate and Fumarate Extend Lifespan in Caenorhabditis elegans.</a> PLoS ONE. 8 (3), 58345 (2013).</li><li>Riddle, D. L., Blumenthal, T., Meyer, B. J., Priess, J. R., Riddle, D. L., Blumenthal, T., Meyer, B. J., Priess, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> C. elegans II. , (1997).</li><li>Churgin, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+imaging+of+Caenorhabditis+elegans+in+a+microfabricated+device+reveals+variation+in+behavioral+decline+during+aging.">Longitudinal imaging of Caenorhabditis elegans in a microfabricated device reveals variation in behavioral decline during aging.</a> eLife. 6, 26652 (2017).</li><li>Scholz, M., Lynch, D. J., Lee, K. S., Levine, E., Biron, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+scalable+method+for+automatically+measuring+pharyngeal+pumping+in+C.+elegans.">A scalable method for automatically measuring pharyngeal pumping in C. elegans.</a> J Neurosci Methods. 274, 172-178 (2016).</li><li>Scholz, M., Dinner, A. R., Levine, E., Biron, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochastic+feeding+dynamics+arise+from+the+need+for+information+and+energy.">Stochastic feeding dynamics arise from the need for information and energy.</a> Proc Natl Acad Sci U S A. 114 (35), 9261-9266 (2017).</li></ol>

作者没有什么可透露的。

微流控工具为研究蠕虫提供了多种好处。与标准的 NGM 琼脂平板相比, 在一台聚甲基硅器件中成像具有更高的成像质量。多个图像可以从一个单一的蠕虫, 与传统的方法, 在这些动物是从板块采摘和安装在显微镜幻灯片的成像。此外, 蠕虫所驻留的微环境可以保持恒定或根据需要进行调制, 允许在环境的构成和动物的反应之间进行精确的映射。
在这里, 我们演示了如何通过一个自...

环境条件的变化可能导致基因程序的激活, 同时诱导和抑制特定基因的表达1,2。这些动态变化可能是不同的组织之间的相同的动物和动物之间。因此, 对这种基因计划的研究要求采用允许对个别动物进行纵向成像的方法, 并提供精确的动态控制环境条件。
近年来, microfabricated 射流装置已被用来研究小动物的反应和行为的许多方面, 包括蠕虫, 苍蝇, 水熊和更多的3,4,5,6, 7。例如, 应用包括深分型、optogenetic 记录神经元活动以响应化学刺激和跟踪马达行为, 如运动和泵浦 8, 9, 10,11。
基于微流控的方法拥有许多性能, 可以帮助对环境线索的长期纵向成像, 包括对局部微环境的精确动态控制, 灵活的设计, 使维护单独的动物在分开的处所和有利属性为成像。然而, 在微流控腔中维持动物很长一段时间, 对他们的良好生命有最小的不利影响是一个挑战, 这需要特别小心的设计, 微流控装置和在执行实验。
在这里, 我们演示了使用 WormSpa, 一个微流控装置的纵向成像的秀丽线虫。5单个蠕虫被限制在腔室中。恒定的低流量的液体和细菌悬浮, 保证蠕虫是良好的喂养和足够活跃, 以保持良好的健康和缓解压力, 和结构的商会允许蠕虫产卵。WormSpa 的设计和操作的简单性应该允许研究人员没有在微流体的经验, 把这一装置纳入他们自己的研究计划。

<table border="0" cellpadding="0" cellspacing="0" style="width:1012px;" width="1011">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">WormSpa</td>
			<td style="width:123px;">N/A</td>
			<td style="width:344px;">N/A</td>
			<td style="width:329px;">The CAD file for WormSpa is available from the Levine lab.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Compound Microscope</td>
			<td>Zeiss</td>
			<td>AxioObserver Z1</td>
			<td>An inverted fluorescence microscope with a motorized stage</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Syringe Pump</td>
			<td>New Era Pump Systems</td>
			<td>NE-501</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tubing</td>
			<td>SCI Scientific Commodities Inc.</td>
			<td>BB31695-PE/5</td>
			<td>0.034&#8221; (0.86 mm) I.D. x 0.052&#8221; (1.32 mm) O.D</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Syringe Tip</td>
			<td>CMLsupply</td>
			<td>901-20-050</td>
			<td>20 Gauge x 1/2&#8221; blunt tip stainless steel canula</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Syringe Filter</td>
			<td>PALL</td>
			<td>4650</td>
			<td>Acrodisc 32 mm Syringe Filter with 5 um Supor Membrane</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Syringe</td>
			<td>Qosina</td>
			<td>C3307</td>
			<td>10 mL Male Luer Lock Syringe</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">3 Way Valve</td>
			<td>ColeParmer</td>
			<td>FF-30600-23</td>
			<td>Large-bore 3-way, male-lock, stopcocks, 10/pack, Non-sterile</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dowel Pin</td>
			<td>McMaster-Carr</td>
			<td>90145A317</td>
			<td>18-8 Stainless Steel Dowel Pins (1/32&#34; Dia. x 1/2&#34; Lg.)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Low Binding Microcentrifuge Tube</td>
			<td style="width:123px;">Corning</td>
			<td>CL S3206</td>
			<td>0.65 mL low binding snap cap microcentrifuge tube</td>
		</tr>
	</tbody>
</table>

a microfluidic platform for longitudinal imaging in caenorhabditis elegans

在过去十年中, 微流控技术已被应用于研究小动物, 包括线虫秀丽线虫, 并证明是有用的, 作为一个方便的实时成像平台提供能力, 精确控制实验条件的实时性。在本文中, 我们演示了使用 WormSpa, 以前发布的自定义微流控装置的单个蠕虫的实时成像。在该装置中, 多个蠕虫单独局限于单独的分庭, 允许多路纵向监测各种生物过程。为了说明这种能力, 我们进行了实验验证, 其中蠕虫感染了致病细菌, 并连续监测免疫应答基因和产卵的表达动态动物。该设备的简单设计和操作使其适合于以往没有经验的微流控实验的用户。我们建议, 这种方法将是有益的, 许多研究人员感兴趣的纵向观察生物过程在明确的条件下。

年龄同步的年轻成人蠕虫 (46 小时后 L1 幼虫拘捕在25°c)12已加载到设备中, 如协议中所述。蠕虫单独位于单独的渠道, 使动物对病原体的反应进行纵向测量。当实验成功时, 大多数蠕虫在实验期间仍留在其通道中。在这种情况下, 单个蠕虫的图像只是通过将其通道放置在成像路径中, 从而避免了主动定位该动物的需要。图 2A</strong...

Watch this Scientific Journal Video about A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans at JoVE.com

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

秀丽线虫纵向成像的微流控平台

在本文中, 我们演示了使用自定义微流控装置的单个蠕虫的实时成像。在该装置中, 多个蠕虫单独局限于单独的分庭, 允许多路纵向监测各种生物过程。

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

a-microfluidic-platform-for-longitudinal-imaging-caenorhabditis

Research

JoVE Journal

Bioengineering

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

6.4K 次观看.  Kongju National University. 在本文中, 我们演示了使用自定义微流控装置的单个蠕虫的实时成像。在该装置中, 多个蠕虫单独局限于单独的分庭, 允许多路纵向监测各种生物过程。

视频: A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

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.

简单的微流控芯片的<em在体内</em&gt;成像<em&gt; C.线虫</em&gt;<em&gt;果蝇</em&gt;和斑马鱼

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

科研

生物工程

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

利用微流控平台的高通量蛋白质表达发生器

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

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.

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 ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

电池挤压作为一种强大的，微流控细胞内传送平台

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

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 physiological dcEF in guiding cell movement during embryo development, cell differentiation, and wound healing has been demonstrated in many studies. By applying microfluidic chips to an electrotaxis assay, the investigation process is shortened and experimental errors are minimized. In recent years, microfluidic devices made of polymeric substances (e.g., polymethylmethacrylate, PMMA, or acrylic) or polydimethylsiloxane (PDMS) have been widely used in studying the responses of cells to electrical stimulation. However, unlike the numerous steps required to fabricate a PDMS device, the simple and rapid construction of the acrylic micro&#64258;uidic chip makes it suitable for both device prototyping and production. Yet none of the reported devices facilitate the efficient study of the simultaneous chemical and dcEF effects on cells. In this report, we describe our design and fabrication of an acrylic-based multichannel dual-electric-field (MDF) chip to investigate the concurrent effect of chemical and electrical stimulation on lung cancer cells. The MDF chip provides eight combinations of electrical/chemical stimulations in a single test. The chip not only greatly shortens the required experimental time but also increases accuracy in electrotaxis studies.

Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

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 ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

高通量单细胞的分离和文化的微流体平台

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

与复用电子检测的粒子的空间跟踪微流体平台

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform

Root hairs increase root surface area for better water uptake and nutrient absorption by the plant. Because they are small in size and often obscured by their natural environment, root hair morphology and function are difficult to study and often excluded from plant research. In recent years, microfluidic platforms have offered a way to visualize root systems at high resolution without disturbing the roots during transfer to an imaging system. The microfluidic platform presented here builds on previous plant-on-a-chip research by incorporating a two-layer device to confine the Arabidopsis thaliana main root to the same optical plane as the root hairs. This design enables the quantification of root hairs on a cellular and organelle level and also prevents z-axis drifting during the addition of experimental treatments. We describe how to store the devices in a contained and hydrated environment, without the need for fluidic pumps, while maintaining a gnotobiotic environment for the seedling. After the optical imaging experiment, the device may be disassembled and used as a substrate for atomic force or scanning electron microscopy while keeping fine root structures intact.

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform

This article demonstrates how to culture Arabidopsis thaliana seedlings in a two-layer microfluidic platform that confines the main root and root hairs to a single optical plane. This platform can be used for real-time optical imaging of fine root morphology as well as for high-resolution imaging by other means.

影像学在两层微流控平台拟南芥幼苗的根头发形态

Root hairs increase root surface area for better water uptake and nutrient absorption by the plant. Because they are small in size and often obscured by ...

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing &gt;50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.

Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing &gt;50,000 single-cell genomes from diverse cell populations.

单细胞基因组测序的超高通量微流控平台

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...

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.

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

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

秀丽线虫纵向成像的微流控平台

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