Name: a microfluidic platform for longitudinal imaging in caenorhabditis elegans
Uploaded: 2018-05-02T13:30:00.000+00:00
Duration: 9 min
Description: Watch this Scientific Journal Video about A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans at JoVE.com

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>

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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><tbody><tr><td>WormSpa</td><td>N/A</td><td>N/A</td><td>The CAD file for WormSpa is available from the Levine lab.</td></tr><tr><td>Compound Microscope</td><td>Zeiss</td><td>AxioObserver Z1</td><td>An inverted fluorescence microscope with a motorized stage</td></tr><tr><td>Syringe Pump</td><td>New Era Pump Systems</td><td>NE-501</td><td></td></tr><tr><td>Tubing</td><td>SCI Scientific Commodities Inc.</td><td>BB31695-PE/5</td><td>0.034&amp;rdquo; (0.86 mm) I.D. x 0.052&amp;rdquo; (1.32 mm) O.D</td></tr><tr><td>Syringe Tip</td><td>CMLsupply</td><td>901-20-050</td><td>20 Gauge x 1/2&amp;rdquo; blunt tip stainless steel canula</td></tr><tr><td>Syringe Filter</td><td>PALL</td><td>4650</td><td>Acrodisc 32 mm Syringe Filter with 5 um Supor Membrane</td></tr><tr><td>Syringe</td><td>Qosina</td><td>C3307</td><td>10 mL Male Luer Lock Syringe</td></tr><tr><td>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><td>Dowel Pin</td><td>McMaster-Carr</td><td>90145A317</td><td>18-8 Stainless Steel Dowel Pins (1/32&quot; Dia. x 1/2&quot; Lg.)</td></tr><tr><td>Low Binding Microcentrifuge Tube</td><td>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

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 ...

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous system can be observed, with single cell resolution, in vivo. Here, we present an integrated protocol for the examination of neurodevelopment in C. elegans embryos. Our protocol combines imaging, lineaging and neuroanatomical tracing of single cells in developing embryos. We achieve long-term, four-dimensional (4D) imaging of living C. elegans&#160;embryos with nearly isotropic spatial resolution through the use of Dual-view Inverted Selective Plane Illumination Microscopy (diSPIM). Nuclei and neuronal structures in the nematode embryos are imaged and isotropically fused to yield images with resolution of ~330 nm in all three dimensions. These minute-by-minute high-resolution 4D data sets are then analyzed to correlate definitive cell-lineage identities with gene expression and morphological dynamics at single-cell and subcellular levels of detail. Our protocol is structured to enable modular implementation of each of the described steps and enhance studies on embryogenesis, gene expression, or neurodevelopment.

Here, we present a combinatorial approach using high-resolution microscopy, computational tools, and single-cell labeling in living C. elegans embryos to understand single cell dynamics during neurodevelopment.

亚细胞分辨率的各向同性光片显微镜及细胞自动动力学分析

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous ...

发育生物学

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One challenge is that embryogenesis dynamically unfolds over a period of about 13 h; this half day-long timescale has constrained the scope of experiments by limiting the number of embryos that can be imaged. Here, we describe a semi-high-throughput protocol that allows for the simultaneous 3D time-lapse imaging of development in 80&#8211;100 embryos at moderate time resolution, from up to 14 different conditions, in a single overnight run. The protocol is straightforward and can be implemented by any laboratory with access to a microscope with point visiting capacity. The utility of this protocol is demonstrated by using it to image two custom-built strains expressing fluorescent markers optimized to visualize key aspects of germ-layer specification and morphogenesis. To analyze the data, a custom program that crops individual embryos out of a broader field of view in all channels, z-steps, and timepoints and saves the sequences for each embryo into a separate tiff stack was built. The program, which includes a user-friendly graphical user interface (GUI), streamlines data processing by isolating, pre-processing, and uniformly orienting individual embryos in preparation for visualization or automated analysis. Also supplied is an ImageJ macro that compiles individual embryo data into a multi-panel file that displays maximum intensity fluorescence projection and brightfield images for each embryo at each time point. The protocols and tools described herein were validated by using them to characterize embryonic development following knock-down of 40 previously described developmental genes; this analysis visualized previously annotated developmental phenotypes and revealed new ones. In summary, this work details a semi-high-throughput imaging method coupled with a cropping program and ImageJ visualization tool that, when combined with strains expressing informative fluorescent markers, greatly accelerates experiments to analyze embryonic development.

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80&#8211;100 C. elegans embryos in a single overnight run. Additionally, image processing and visualization tools are included to streamline data analysis. The combination of these methods with custom reporter strains enables detailed monitoring of embryogenesis.

半高通量成像方法和数据可视化工具包，用于分析C. elegans胚胎发育

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One ...

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Imaging behavior and neural activity over long time scales without immobilization of the animal is a prerequisite to understand behavior. Agarose microfluidic chambers imaging (AMI) can be used to image neural activity and behavior for all life stages of Caenorhabditis elegans.

琼脂糖微室的长期钙成像<em&gt;秀丽隐杆线虫</em

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

神经科学

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples1,2. Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage3, thus providing an ideal experiment model for studying questions in cell biology4,5and development6-9. C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis10,11) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis12-15). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters16,17. These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo18-21. In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.

成像 C。线虫胚胎使用啶显微镜和开放源码软件

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

生物学

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

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

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

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

应用一种适应微流控嗅觉芯片的神经活性成像对雄性线虫神经元的信息素反应

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

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

One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the influence of mechanical stress on cellular function is still poorly understood. In part, this knowledge gap exists because few tools enable simultaneous deformation of tissue and cells, imaging of cellular activity in live animals, and efficient restriction of motility in otherwise highly mobile model organisms, such as the nematode Caenorhabditis elegans. The small size of C. elegans makes them an excellent match to microfluidics-based research devices, and solutions for immobilization have been presented using microfluidic devices. Although these devices allow for high-resolution imaging, the animal is fully encased in polydimethylsiloxane (PDMS) and glass, limiting physical access for delivery of mechanical force or electrophysiological recordings. Recently, we created a device that integrates pneumatic actuators with a trapping design that is compatible with high-resolution fluorescence microscopy. The actuation channel is separated from the worm-trapping channel by a thin PDMS diaphragm. This diaphragm is deflected into the side of a worm by applying pressure from an external source. The device can target individual mechanosensitive neurons. The activation of these neurons is imaged at high-resolution with genetically-encoded calcium indicators. This article presents the general method using C. elegans strains expressing calcium-sensitive activity indicator (GCaMP6s) in their touch receptor neurons (TRNs). The method, however, is not limited to TRNs nor to calcium sensors as a probe, but can be expanded to other mechanically-sensitive cells or sensors.

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

New tools for mechanobiology research are needed to understand how mechanical stress activates biochemical pathways and elicits biological responses. Here, we showcase a new method for selective mechanical stimulation of immobilized animals with a microfluidic trap allowing high-resolution imaging of cellular responses.

利用微流体装置进行机械刺激和高分辨率成像的研究C.线虫

One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the ...

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

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

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

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

用于秀丽隐杆线虫长期生长和成像的简单微流控芯片

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

Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions

Caenorhabditis elegans are widely used to study aging biology. The standard practice in C. elegans aging studies is to culture groups of worms on solid nematode growth media (NGM), allowing the efficient collection of population-level data for survival and other physiological phenotypes, and periodic sampling of subpopulations for fluorescent biomarker quantification. Limitations to this approach are the inability to (1) follow individual worms over time to develop age trajectories for phenotypes of interest and (2) monitor fluorescent biomarkers directly in the context of the culture environment. Alternative culture approaches use liquid culture or microfluidics to monitor individual animals over time, in some cases including fluorescence quantification, with the tradeoff that the culture environment is contextually distinct from solid NGM. The WorMotel is a previously described microfabricated multi-well device for culturing isolated worms on solid NGM. Each worm is maintained in a well containing solid NGM surrounded by a moat filled with copper sulfate, a contact repellent for C. elegans, allowing longitudinal monitoring of individual animals. We find copper sulfate insufficient to prevent worms from fleeing when subjected to aversive interventions common in aging research, including dietary restriction, pathogenic bacteria, and chemical agents that induce cellular stress. The multi-well devices are also molded from polydimethylsiloxane, which produces high background artifacts in fluorescence imaging. This protocol describes a new approach for culturing isolated roundworms on solid NGM using commercially available polystyrene microtrays, originally designed for human leukocyte antigen (HLA) typing, allowing the measurement of survival, physiological phenotypes, and fluorescence across the lifespan. A palmitic acid barrier prevents worms from fleeing, even in the presence of aversive conditions. Each plate can culture up to 96 animals and easily adapts to a variety of conditions, including dietary restriction, RNAi, and chemical additives, and is compatible with automated systems for collecting lifespan and activity data.

Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions

Here, we present a protocol to culture isolated individual nematodes on solid media for lifelong physiological parameter tracking and fluorescence quantification. This culture system includes a palmitic acid barrier around single-worm wells to prevent animals from fleeing, allowing the use of aversive interventions, including pathogenic bacteria and chemical stressors.

在固体培养基上长期培养单个 秀丽隐杆线虫 ，用于纵向荧光监测和厌恶干预

Caenorhabditis elegans are widely used to study aging biology. The standard practice in C. elegans aging studies is to culture groups of worms on solid ...

秀丽线虫纵向成像的微流控平台

摘要

探索更多视频

此视频中的章节

简单的微流控芯片的

基于微流体Electrotaxis的，为按需定量分析

亚细胞分辨率的各向同性光片显微镜及细胞自动动力学分析

半高通量成像方法和数据可视化工具包，用于分析C. elegans胚胎发育

琼脂糖微室的长期钙成像

成像 C。线虫胚胎使用啶显微镜和开放源码软件

应用一种适应微流控嗅觉芯片的神经活性成像对雄性线虫神经元的信息素反应

利用微流体装置进行机械刺激和高分辨率成像的研究C.线虫

用于秀丽隐杆线虫长期生长和成像的简单微流控芯片

在固体培养基上长期培养单个秀丽隐杆线虫，用于纵向荧光监测和厌恶干预

秀丽线虫纵向成像的微流控平台

摘要

探索更多视频

此视频中的章节

简单的微流控芯片的

基于微流体Electrotaxis的，为按需定量分析

亚细胞分辨率的各向同性光片显微镜及细胞自动动力学分析

半高通量成像方法和数据可视化工具包，用于分析C. elegans胚胎发育

琼脂糖微室的长期钙成像

成像 C。线虫胚胎使用啶显微镜和开放源码软件

应用一种适应微流控嗅觉芯片的神经活性成像对雄性线虫神经元的信息素反应

利用微流体装置进行机械刺激和高分辨率成像的研究C.线虫

用于秀丽隐杆线虫长期生长和成像的简单微流控芯片

在固体培养基上长期培养单个 秀丽隐杆线虫 ，用于纵向荧光监测和厌恶干预

在固体培养基上长期培养单个秀丽隐杆线虫，用于纵向荧光监测和厌恶干预