Name: agarose microchambers for long-term calcium imaging of caenorhabditis elegans
Uploaded: 2015-06-24T15:00:00.000+00:00
Duration: 7 min 51 s
Description: Watch this Scientific Journal Video about Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans at JoVE.com

马普学会和哥廷根研究生院少年组助学金资助HB这项工作。

1，仪器仪表，文化传媒，和菜<ol><li>使用显微镜，其能够保持在焦点样品并配有一个自动台架。构建定制盖加热器或购买商业解决方案。设置LED的EMCCD摄像系统，其中，照相机的曝光使用制造商的说明触发器LED照明通过TTL信号。 注:见讨论的细节。 </li><li>聚二甲基硅氧烷（PDMS）邮票: <ol><li>制作PDMS邮票在微流体设备或者由商业代工生产的PDMS邮票。使用商业代工，发送一个AutoCAD文件到公司指定设备的深度（15微米，例如）。从交付代工后，切PDMS芯片到使用手术刀16个邮票。 </li><li>结合每个邮票利用空气等离子载玻片。粘接，露出两个PDMS压模和载玻片在空气等离子体中一回合1分钟（使用最高血浆设置在0.5毫巴）。确保在等离子处理的表面粘接朝上。然后，将PDMS压模到玻璃载玻片上。 </li></ol></li><li>取3.5公分底部盘和切出的18×18毫米的正方形区域由使用立式铣床和尖锐旋转切割盘的底部的中心。使用切割器和缓慢进给的低旋转速度，以防止塑料熔化由于由摩擦产生的热量。准备几盘一次。 注:底部盘可以在实验后重复使用多次，如果它被浸泡在纯乙醇O / N后清洗。 </li><li>琼脂糖: <ol><li>溶解3克高熔点琼脂糖在100毫升的S-基底（5.85克氯化钠，将1g K 2 HPO 4，6克KH 2 PO 4，将1ml胆固醇（5毫克/毫升在乙醇中），H 2 O至1 L时，通过高压灭菌）通过煮沸。使的溶解琼脂糖等份在2ml Eppendorf管中并保存位置即另外，准备一个批次低熔点琼脂糖的完全相同的方式作为高熔点琼脂糖。 </li><li>在使用之前，放置高熔点的3等份琼脂糖到一个加热块在95 - 98℃。在使用之前，放置低熔点的一个等份琼脂糖上的加热块在95 - 35℃ - 98℃，直到它已在30熔融，然后到一个加热块。 </li></ol></li></ol> 2.选择动物<ol><li>生长在蠕虫对种子NGM板低密度获得洁净的动物。确保有充足的食物，只有少数动物的上盘。 </li><li>对于成像L1幼虫，转移约30鸡蛋含有胚胎的饼干阶段到新鲜种子NGM板。对于转移后幼虫或成虫C.线虫，转移约30蠕虫到新鲜种子NGM板。 </li></ol> 3.准备琼脂糖微容器<ol><li>准备的菜: <ol><li>取的塑料培养皿在底部的正方形开口的18×18毫米，其放置朝上开口倒置。通过将一块20×20mm的双面胶带上的开口封闭开口。转动盘周围，使得胶带在底部和放置盘到硬表面上。切免费使用手术刀开幕。 </li><li>用吸管P1000，填补加入2ml的S-基础3％高熔点琼脂糖入菜。将琼脂糖到周围的开口面积，让凝固。等待，直到琼脂糖是坚实的。 </li><li>扭转盘和剥离覆盖双面胶带，以使粘侧将保持上盘和将暴露的保护膜。 注意:作为一个结果，粘性胶带的一个细环将环绕该开口的外部。琼脂糖用作湿气贮存稍后将环绕样本。 </li></ol></li><li>铸造微容器: <ol><li>揭露日ËPDMS表面与空气等离子成型为20 - 60秒。 注意:此等离子体处理使该PDMS表面的亲水性，从而防止气泡的俘获，产生更清晰的印刷品。 </li><li>构建高度相等两个间隔件通过层叠5 - 9载玻片上。的地方，在平行于它们的长边，在第一隔离片叠，然后单个载玻片，然后再一个垫片栈。放置一个包含PDMS压模正交跨越衬垫的载玻片。调整间隔物的高度，以便有的PDMS压模的模制表面和单个载玻片之间约1.5mm的空间。 </li><li>放置一滴热液体高熔点琼脂糖上邻近的PDMS压模单个载玻片和快速地滑动PDMS压模垂直插入液体琼脂糖。让琼脂糖巩固。检查它得到一个不透明的外观，这通常需要约2分钟。一个垂直移动决绝的印记。 注意:为方便起见，GLUE的间隔用双面胶带一起滑动。印记的垂直运动可以防止气泡被困在琼脂糖。 </li></ol></li><li>与OP50细菌转移鸡蛋或蠕虫到一起用细铂丝挑的琼脂糖。使用睫毛食品分发一个鸡蛋或每室1蠕虫在一起。填补约30鸡蛋到一个琼脂糖垫。 </li><li>切含有填充微容器的琼脂糖切片成约15×15毫米的正方形，使其能装进盘的开口。拿起平方琼脂糖切片用钳子和颠倒其放置到的20×20毫米的玻璃盖玻片。一旦掉线，不要再抬不起来或周围滑动，因为这可能会导致细菌和蠕虫被推出自己的商会。 </li><li>大会的菜: <ol><li>放置玻璃盖玻片上的塑料盘的开口。轻轻按下玻璃盖玻片上环制成的双面胶带。小心不要打破玻璃。 </li><li>转动盘倒置，并使用P1000吸管填补冷却至约30℃的琼脂板含有微容器和琼脂糖贮存有液体低熔点琼脂糖之间的差距。等到琼脂糖凝固。 </li><li>密封有盖培养皿。对于倒置显微镜用热盖。对于正置显微镜使用普通的盖子，用封口膜密封的菜。 </li></ol></li><li>整理微容器的准备后，检查它们在立体显微镜下。正确的填充物是至关重要的。见讨论的细节。 </li></ol> 4.钙成像<ol><li>使用转基因的菌株表达基因编码的钙传感器，如HBR16（goeIs5 [PNMR-1 :: SL1〜GCaMP3.35-SL2 :: UNC-54-3&#39;UTR，UNC-119（+）]）27。 </li><li>使用复合显微镜配备了宽视场落射荧光。该EMCCD相机的TTL输出连接到LED的TTL输入，使得每次照相机记录的帧的样本将被点亮。使用约5毫秒的曝光时间。 300 - 在50范围内使用EM增益。 </li><li>指定突发电影运行24小时，每个蠕虫被成像，每15 - 30分钟首先为20秒，DIC，然后20秒用绿色荧光记录GCaMP，然后取mKate2信号的最终图像被送到控制的表达水平。每个突发时使用的2 /秒的帧速率。 </li><li>对于可视化数据的检查，使用假色图，以提高荧光强度变化小的知名度。积荧光数据如ΔF/女，具有F为荧光的平均基线值。钙数据分析的详细描述可以在文献20中找到。 </li></ol>多虫的5并行AMI <ol><li>将含微容器到显微镜的菜，注重样品和搞autofoCUS。建立一个软件协议以便摄像机获取的突发40的图像帧中20秒，每隔半小时24小时，这将导致每个蜗轮1,920帧，数据的合理量。设置扫描，使​​其使用阶段每个访问虫。目标是在一次运行片约30蠕虫。 </li><li>图片多个蠕虫通过缩小， 即用较低的放大倍率。使用较低的放大倍数，涵盖多个微室的摄像头芯片，同时拍摄几个相邻的微容器。图像获取结束后，通过裁剪的覆盖一只动物感兴趣区域分离为每个单独的室中的数据。 </li><li>快速评估移动数据，使用帧减法28-32。 </li></ol> 6.不同成像C.生活的各个阶段线虫 <ol><li>使用琼脂糖微室为C的所有生命阶段从线虫 L1到成人，包括dauers。使用适当的腔尺寸DIF同的生活阶段被示于表1。 </li></ol><table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td>生命阶段</td><td>炉膛尺寸</td><td>前房深度</td><td>典型录制时间</td><td>放大</td></tr><tr><td> L1 </td><td> 190×190微米</td><td> 10 - 15微米</td><td>鸡蛋 - L2中旬</td><td> 400X </td></tr><tr><td> L2 </td><td> 370 x 370微米</td><td> 15微米</td><td>鸡蛋 - L3 </td><td> 200X </td></tr><tr><td>持久幼虫</td><td> 370 x 370微米</td><td> 25微米</td><td> 4天</td><td> 200X </td></tr><tr><td> L3 </td><td> 700×700微米</td><td> 45微米</td><td> L2 - L4 </td><td> 200X </td></tr><tr><td> L4 </td><td> 700×700微米</td><td> 45微米</td><td>年轻L4 - 年轻成人</td><td> 100X </td></tr><tr><td>年轻的成年</td><td> 700×700微米</td><td> 45微米</td&gt; <td> 12 - 24小时</td><td> 100X </td></tr></tbody></table> 表1.商会大小不同的生命阶段。显示的是 腔的尺寸和放大倍率，这对一个8×8毫米摄影机芯片优化各个阶段非常有用。

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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+dauerlarva,+a+post-embryonic+developmental+variant+of+the+nematode+Caenorhabditis+elegans.">The dauerlarva, a post-embryonic developmental variant of the nematode Caenorhabditis elegans.</a> Developmental Biology. 46, 326-342 (1975).</li><li>Schwarz, J., Lewandrowski, I., 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+activity+of+a+sensory+neuron+during+a+sleep-like+state+in+Caenorhabditis+elegans.">Reduced activity of a sensory neuron during a sleep-like state in Caenorhabditis elegans.</a> Current Biology. 21, R983-R984 (2011).</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, 12-14 (2012).</li><li>Schwarz, J., 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+sleep-like+quiescence+in+both+hyperactive+and+hypoactive+mutants+of+the+Galphaq+Gene+egl-30+during+lethargus+in+Caenorhabditis+elegans.">Reduced sleep-like quiescence in both hyperactive and hypoactive mutants of the Galphaq Gene egl-30 during lethargus in Caenorhabditis elegans.</a> PLoS One. 8, e75853 (2013).</li></ol>

作者宣称，他们有没有竞争的财务权益。

硬件显微镜的焦点长期图像采集期间将典型地漂移。对于复合显微镜，集中保存系统可以从各大显微镜制造商购买。在的情况下的化合物显微镜聚焦控制过普赖西，一个简单的替代方法是使用立体显微镜。复合显微镜允许使用的目标的具有高数值孔径（NA），并可以容易地实现自动化。 40X油目标是非常适合。水浸泡镜片是因为在长期的成像蒸发并不理想。微分干涉对比（DIC）产生很好的对比，有助于后续的形态和行为变化，但简单的明视场成像也可使用。对于DIC或明场成像，通过放置一个红色光过滤器进入显微镜透射照明路径中使用的红光。用于扫描的多个微容器的自动阶段是必要的。表现最好的是交流当一个阶段是使用具有非线性的加速和减速，并可以设置为低扫描速度，以防止在扫描期间扰乱动物hieved。我们没有观察到的行为反应或钙增加扫描期间机械敏感神经元（ALM和PLM），表明慢扫描确实不激活蠕虫的机械敏感系统（数据未显示）。商用LED系统都可以使用。有几家公司提供准备使用的解决方案，包括LED指示灯，在不同的波长。 LED应该有外部触发LED与TTL信号的选项。一个高度敏感的摄像头是需要移动的动物钙成像。 EMCCD相机是市场上最敏感的相机。相机需要有曝光期间一个TTL输出（也称为"火"输出）。的盖加热器是必需的，如果使用倒置显微镜，以防止结露盖子上。上的直立式显微镜，盘子将被放置使得盖子的Wi会位于底部，因此缩合被防止，且没有盖子加热是必需的。该盖应紧紧关闭的盘，以防止在长期成像的水分蒸发。 PDMS邮票包含该结构用于模制琼脂糖PDMS压模的表面面向远离载玻片，它支持PDMS压模。名单与公司提供定制的微流控芯片可以在维基百科上找到（ <a href="http://en.wikipedia.org/wiki/List_of_microfluidics_related_companies" target="_blank">http://en.wikipedia.org/wiki/List_of_microfluidics_related_companies</a> ）。 灌装线虫到他们的室这是在该协议中最关键的步骤和以下几点应考虑:a）琼脂糖的湿润是用于传输蠕虫和成像是至关重要的。如果琼脂糖太潮湿， 即，有一个液体COV化工e圈的几个微容器的区域，这将是不可能的分发细菌食物和蠕虫以受控的方式，因为液体会使细菌流走。如果琼脂糖过于干燥，然后在细菌和蠕虫可以不下车拾取容易，这意味着增加力需要被用于删除蠕虫和细菌容易导致损坏琼脂糖。在很多蠕虫填充时的琼脂糖可能会干涸。在最坏的情况下，将琼脂糖进行到底，该商会将崩溃这么干的。如果琼脂糖过于干燥它可以通过将一小滴的S-基底的（约2微升）到那里有没有蠕虫芯片的侧面被水化。再浸铂丝挑到液体和连拉一些液体进入其中的腔室填充有蠕虫的区域。 B）的食品的量对于成功的长期成像是至关重要的。如果没有足够的食物，蠕虫可能会耗尽它。如果有过多的食物的腔室的腔不会表现得像一个液体而是象固体，并将允许蠕虫从腔室通过推断细菌逃脱。旨在获得菌悬液，填补了整个房间。蠕虫在与琼脂和玻璃表面沿其大部分长度的实验期间保持接触，并出现爬行行为是类似的运动上的板和异种在液体颠簸。 C）机械损坏琼脂糖可以毁掉实验。一个精挑是必不可少的。它不应该有尖角。附连到吸管尖的柔软睫毛可以被用于移动使用铂拾取的细菌或蠕虫进入腔室来代替。在大多数情况下，然而，过干的琼脂糖是损坏的原因。拾取或睫毛应理想情况下，仅勉强触及琼脂糖表面琼脂糖本身和水膜应拉断虫和细菌。当密封室中，再次，琼脂糖的水分是重要的。应该不会是在琼脂糖表面无任何液体，因为这可能会在密封洗去细菌和蠕虫。大的气泡可以通过轻轻提起琼脂板的角被除去。小气泡，比室常常会困在里面的小室。这些气泡是不存在的问题，并会通过吸收消失。组装的菜后，再次检查琼脂糖是不是太干或太湿。腔室应很好地密封，不应该有液体腔室之间的任何流动。如果琼脂糖过于潮湿，商会将无法正确密封。蠕虫可能逃跑或者他们的食物会被冲走。如果样品太潮湿，简单的打开盖子，让琼脂糖干一两分钟。如果琼脂糖过于干燥，商会可以崩溃，蠕虫会逃跑。 通过缩小扩大对于荧光成像，缩小由在较低的放大倍率得到的光的量低的限制。此外，EMCCD摄像机灵敏度优化，并通常具有相对低的分辨率。然而，扩大成像四倍非常可能的。例如，四个腔室190微米×190微米可以使用140X放大倍率（使用20倍的目标和0.7X镜头卡口实现）和512×512像素，8×8毫米摄影机，当适合到一帧。高分辨率的摄像头，大芯片（如SCMOS相机16.6毫米×14毫米芯片2560 x 2560个象素= 5.5万像素）优化扩大DIC和明场成像。例如，高达30的L1蠕虫在190微米×190微米的腔室可以通过使用放大100倍时配合在本相机的每一帧（参见图3）。原则上，缩小和扫描也可以合并，以获得更大数目的动物。大多数的神经元留相当好聚焦，以便只有一个焦平面需要被成像。如果神经元被发现迁出的重点，AZ利用压电驱动器扫描可采取在每个时刻point。 适应不同的行为该协议提供了跨越长时间尺度的行为是个好主意。显然，需要的脉冲串的时序，以适应不同的行为和生命阶段。 该技术的局限性有几个因素限制了成像的持续时间。最重要的是食物的量。一旦食物的消耗，幼虫停止发展。因此，在小室（​​190×190微米）蠕虫发展到了L3的阶段，然后逮捕。如果需要较长的成像时间，较大的腔室必须被使用。长期成像的最大持续时间是在2.5的范围内 - 3天。如果较长的成像是必需的，蠕虫需要回收，并放入新的腔室。当成像成虫，另一限制是由这些蠕虫的后代所引起的。成虫铺设从幼虫孵化的蛋。这些幼虫也将留在室内，消耗食物，可能会干扰图像分析。如果后代是一个问题，一个解决方案是使用无菌成人或反复将蠕虫到新鲜室。以回收蠕虫，含有该室中的琼脂糖板坯切断自由用解剖刀，被拉断盖玻片，并放置到一个NGM板从该蠕虫可以恢复。另一个限制是蠕虫相对小区域的限制。这可能是一个问题，如果远程移动需要分析。而动物在室可机械地和optogenetically 21,27,34,35来刺激，该腔室的密封性质将使其难以适用的水溶性或挥发性兴奋剂。重要的生物气体，如氧气或二氧化碳所用的琼脂自由扩散。大型水库的空气在盘应保持气体浓度室恒定需要实验的时间。然而，应该记住的是，当地氧concentratioN的腔室可以更类似于液体培养基中培养比在平板上找到了条件。 相对于现有方法的意义微流体器件极大地推进行为和发育研究C.线虫 。通常情况下，微流体结构是由PDMS 12。在这里，我们描述了一个协议，用于从琼脂糖制成微文化室。该技术的强度是高的成像质量，具有生理测量，长期成像，以及合理的高吞吐量特性的相关性的组合。高图像质量，通过使用高NA目标通过玻璃盖玻片成像实现的。其结果是，能够进行荧光成像，如钙成像和亚细胞结构的共焦成像。因为这些动物未固定像在其它系统中，它允许与生理测量行为的相关性。 BEC澳洲英语的动物有充足的食物，他们不断开发使长期成像。该系统可以像很多蠕虫在一个跑，因为动物被限制在各自定义室。因此，该方法可以容易地按比例放大9,27,34-36。 未来的应用到目前为止，该系统已主要用于研究睡眠行为的C.线虫幼虫L1。然而，适应的各个阶段将有可能研究广泛行为也在dauers和成人。行为的宽阵列可以研究这种技术从交配到产卵。

Caenorhabditis elegans has been established as a model system to study behavior1. Due to its amenability to genetics, the molecular and cellular mechanisms underlying behavior can be studied. Many behaviors occur on long time scales. Two principal approaches can be used to observe motile animals over long time scales. The first approach is following the animal during movement using an automated stage or camera2-8 and the second is to restrict the movement to a range that is at least as small as the field of view of the camera9-14. Both methods have their advantages. Tracking allows following an animal over long spatial ranges but limits the number of animals that can be discerned in one experiment. Restricting the movement of the animals allows scaling up the observation to many individuals at the same time by using arrays of restricted compartments. 
Because the nematode is transparent, live fluorescent imaging can be performed non-invasively15. Calcium imaging provides a functional readout for the activity of excitable cells and is established for C. elegans16-20. Calcium enters the cell via channels in the plasma membrane that open upon depolarization. Thus, calcium acts as a proxy for neural activity. Calcium sensors can be grouped in two major classes, ratiometric and non-ratiometric sensors. Both classes employ conformational changes of calcium-binding proteins induced upon binding of calcium. Ratiometric sensors contain two fluorescent proteins. When the lower-wavelength fluorescent protein is excited, a part of the light energy is transmitted to the higher wavelength fluorescent protein as a function of their distance in a process called Fluorescence Resonance Energy Transfer or F&#246;rster Resonance Energy Transfer (FRET)21. Non-ratiometric sensors are based on circularly permuted GFP and employ de-quenching of the fluorophore caused by calcium binding22. Each class has its advantages. While ratiometric sensors are less sensitive to movement or expression artifacts, non-ratiometric sensors typically have a higher dynamic range. Both ratiometric and non-ratiometric sensors have been useful to study the activity of excitable cells in C. elegans16-20,23,24. 
When doing long-term fluorescence imaging, the experimenter will have to deal with several potential challenges: 1. Disturbance of behavior through excitation light: Worms are sensitive to short-wavelength fluorescence excitation light and avoid light from the violet to blue range that is used for calcium imaging25,26. Worms respond with either a backward or a forward escape response25,26. Thus, the amount of light needs to be controlled. 2. Bleaching of the fluorescent sensor protein: Often, bleaching of the fluorescent protein hampers long-term imaging. Typically, however, light intensities that are needed to observe bleaching are higher than the light intensities that cause disturbing effects on the behavior of the animals. Thus, bleaching is only a theoretical problem in this type of calcium imaging. 3. Worms inside the microchambers are not fixed and move constantly during wake behavior and images may appear blurred if the worm is not immobilized. All these challenges can be solved using extremely short exposure times with low light intensity. This can be realized by using a highly sensitive EMCCD camera with short exposure times and external triggering of a light emitting diode (LED). To expose the worms to the illumination light as briefly as possible (only during the exposure time of the camera) the LED is externally triggered using a transistor-transistor logic (TTL) signal that the camera emits during exposure resulting in an illumination of the worm for precisely the time the camera chip is exposed. This also means that the EMCCD chip will be dark during data readout, which is optimal for readout performance of this chip. 
Previously, agarose microchambers have been developed for long-term fluorescence imaging of C. elegans larvae9. Here, it is described how agarose micro-chambers can be used for long-term calcium imaging for any life stage of C. elegans, how calcium imaging can be performed, and how this method can be upscaled to assay many individual worms in parallel.

<table><tbody><tr><td>High melting point agarose</td><td>Fisher Bioreagents</td><td>BP(E)164-1000</td><td></td></tr><tr><td>Top Vision low melting point agarose</td><td>Thermo Scientific</td><td>R0801</td><td></td></tr><tr><td>LED system</td><td>CoolLED</td><td>pE-2</td><td></td></tr><tr><td>Compound microscope</td><td>Nikon</td><td>TiE</td><td></td></tr><tr><td>Stereomicroscope</td><td>Leica</td><td>M5</td><td></td></tr><tr><td>EMCCD camera</td><td>Andor</td><td>iXon 897</td><td>now sold as iXon Ultra 897</td></tr><tr><td>sCMOS camera</td><td>Andor</td><td>Neo</td><td></td></tr><tr><td>Plasma cleaner</td><td>Harrick</td><td>PDC-23G2</td><td></td></tr><tr><td>Lid heater</td><td>MPI workshop</td><td>custom made</td><td></td></tr><tr><td>Plastic dish</td><td>Falcon</td><td>08-757-100A</td><td></td></tr><tr><td>Z-stage for the microscope</td><td>Prior</td><td>Nano Scan Z</td><td></td></tr><tr><td>X-Y stage for the microscope</td><td>Prior</td><td>Proscan III</td><td></td></tr><tr><td>PDMS stamp</td><td>Abbott Laboratories</td><td>custom made</td><td></td></tr><tr><td>Red light filter</td><td>Chroma</td><td>ET660/50m</td><td></td></tr><tr><td>35 mm Petri dish</td><td>Falcon</td><td>Falcon Disposable Petri Dishes, Sterile, Corning</td><td></td></tr><tr><td>Double sided sticky tape</td><td>Sellotape/Henkel</td><td>double sided 25 mm x 33 m</td><td>alternatively advance double sided sticky tape from Rubance Adhesives can be used.</td></tr></tbody></table>

agarose microchambers for long-term calcium imaging of caenorhabditis elegans

行为是由神经系统控制。钙成像是在透明线虫一个简单的方法秀丽隐杆线虫中的各种行为来衡量神经元的活动。关联神经活动与行为，动物不应被固定，但应该能够移动。在很长一段时间尺度发生许多行为的变化和需要记录多个小时的行为。这也使得有必要培养在食物存在的蠕虫。如何才能蠕虫培养和他们的神经活动在成像长时间尺度？琼脂糖微腔成像（AMI）以前开发的文化，观察小幼虫，现在已经适应了研究，从早期的L1所有生命阶段，直到C的成年阶段线虫 。 AMI可以在C.不同生命阶段进行线虫 。长期钙成像是通过不使用短外部触发曝光COM的固定实现动物bined与电子倍增电荷耦合器件（EMCCD）相机记录。缩小或扫描可以将此方法扩展到图像上并行40蠕虫。因此，一种方法是在C的所有生命阶段来描述图像的行为和神经活动在较长的时间尺度线虫 。

琼脂糖微容器可以适用于C的任何生命阶段线虫 。如在图1A中可以看出，在此期间的L1 lethargus幼体发育和睡眠行为可以被观察到。显示的是室尺寸是190×190微米，10微米深。如果较长的时间尺度是必需的，较大的腔室都可以使用。如可以看到的， 如图1B所示 ，使用的腔室具有较大尺寸（370 x 370微米，25微米深）允许C的发展从线虫到鸡蛋成人。 图1C显示了使用在室内种植，从卵到成虫蠕虫的框架减法行为的长期变化进行分析。显示的是平均强度框架减法后的阵阵选择唤醒和lethargus期间。下部后帧减法数值的均值强度，蠕虫的流动性越低。显示从一个唤醒状态，每幼虫阶段一lethargus条件所选的突发痕迹。该在开发过程中的平均强度观测增加主要是由于动物的增加对比度和尺寸。 图1D示出了持久幼虫，它们是在不利的环境条件33啮合的替代生命阶段。腔尺寸为370 x 370微米，15微米深。需要注意的是持久幼虫放入该室无细菌的食物，这将导致从持久幼虫阶段出口。 图1E示出了一个已经铺设许多鸡蛋到室成虫。用于成人腔尺寸为700×700微米，45微米深。最后， 图1F示出了一个成人雌雄同体和阳配合成人室内部。 在游动的蠕虫钙成像是可能的GCaMP钙传感器。 图2A，命令interneuron AVA为L1幼虫27的B显示钙成像。 图2C，D表示活性钙为sa我在成年动物类型的神经元。 扩大长期成像是可能通过扫描和由缩小图3A示出的30蠕虫在一个摄像头芯片同时成像的具有500万像素的照相机的图像质量。 图3B示出的4蠕虫钙同时拍摄到的图像的质量。 <img alt="图1" src="/files/ftp_upload/52742/52742fig1highres.jpg" width="700" /> 图1.急性心肌梗死适应C.所有生命阶段线虫 。（A）幼虫从早期L1成像直到L2初期阶段，190×190微米室。 （ 二）从卵发育到成虫在370 x 370微米室。使用框架减法蠕虫（三）流动性评估。示出的是在图像的帧相减后的所有图片一所选的突发电影的每个幼虫STA的平均强度GE唤醒和lethargus行为。 （D）一种持久幼虫在没有食物在370 x 370微米室。 （E）成人雌雄同体有许多产卵成700×700微米室。 （F）男性的交配和一个700×700微米室内的雌雄同体。 YA意味着年轻的成年人。 <a href="https://www.jove.com/files/ftp_upload/52742/52742fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52742/52742fig2highres.jpg" width="700" /> 图2.钙成像的AMI。（A）的钙成像GCaMP3在命令interneuron使用核磁共振-1启动子的L1幼虫AVA。显示有两个假彩色图像。在左图像，AVA的活性低和蜗杆不使一个向后运动。在右边的图像AVA的活动<html>高，蠕虫向后移动。 （B）钙瞬变随时间的L1蠕虫病毒。 （C，D）在成年动物钙瞬变。 <a href="https://www.jove.com/files/ftp_upload/52742/52742fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52742/52742fig3highres.jpg" width="700" /> 图3.成像蠕虫的多个并行通过放大出来。（A）采用190×190微米室每帧30 L1蠕虫同时成像和100X放大倍率（使用10X物镜）和16.6×14毫米相机的芯片。 （ 二）采用370 x 370微米室和100倍的放大倍率和16.6×14毫米相机的芯片感兴趣的区域每帧4 L3蠕虫同时成像。等="_空白"&gt;点击此处查看该图的放大版本。

Watch this Scientific Journal Video about Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans at JoVE.com

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 ...

agarose-microchambers-for-long-term-calcium-imaging-caenorhabditis

Nanyang Technological University

Research

JoVE Journal

Neuroscience

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

9.5K 次观看.  Max Planck Institute for Biophysical Chemistry. 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. 

视频: Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for the targeted expression of genetically encoded calcium sensors. This protocol outlines the use of these sensors for the in vivo imaging of calcium dynamics in targeted cells, specifically the body wall muscles of the worms. By utilizing the co-expression of presynaptic channelrhodopsin, stimulation of acetylcholine release from excitatory motor neurons can be induced using blue light pulses, resulting in muscle depolarization and reproducible changes in cytoplasmic calcium levels. Two worm immobilization techniques are discussed with varying levels of difficulty. Comparison of these techniques demonstrates that both approaches preserve the physiology of the neuromuscular junction and allow for the reproducible quantification of calcium transients. By pairing optogenetics and functional calcium imaging, changes in postsynaptic calcium handling and homeostasis can be evaluated in a variety of mutant backgrounds. Data presented validates both immobilization techniques and specifically examines the roles of the C. elegans sarco(endo)plasmic reticular calcium ATPase and the calcium-activated BK potassium channel in the body wall muscle calcium regulation.

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

This method provides a way to couple optogenetics and genetically encoded calcium sensors to image baseline cytosolic calcium levels and changes in evoked calcium transients in the body wall muscles of the model organism C. elegans.

在 Vivo 钙成像中，在 C. elegans身体壁肌肉

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for ...

科研

生物化学

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

C.在体内神经元钙成像技术线虫

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

神经科学

Calcium Imaging in Freely Behaving Caenorhabditis elegans with Well-Controlled, Nonlocalized Vibration

Nonlocalized mechanical forces, such as vibrations and acoustic waves, influence a wide variety of biological processes from development to homeostasis. Animals cope with these stimuli by modifying their behavior. Understanding the mechanisms underlying such behavioral modification requires quantification of neural activity during the behavior of interest. Here, we report a method for calcium imaging in freely behaving Caenorhabditis elegans with nonlocalized vibration of specific frequency, displacement, and duration. This method allows the production of well-controlled, nonlocalized vibration using an acoustic transducer and quantification of evoked calcium responses at single-cell resolution. As a proof of principle, the calcium response of a single interneuron, AVA, during the escape response of C. elegans to vibration is demonstrated. This system will facilitate understanding of neural mechanisms underlying behavioral responses to mechanical stimuli.

Calcium Imaging in Freely Behaving Caenorhabditis elegans with Well-Controlled, Nonlocalized Vibration

Reported here is a system for calcium imaging in freely behaving Caenorhabditis elegans with well-controlled, nonlocalized vibration. This system allows researchers to evoke nonlocalized vibrations with well-controlled properties at nano-scale displacement and to quantify calcium currents during responses of C. elegans to the vibrations.

自由行为 秀丽隐杆线虫 的钙成像，具有良好控制，非局部化振动

Nonlocalized mechanical forces, such as vibrations and acoustic waves, influence a wide variety of biological processes from development to homeostasis. ...

行为学

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

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

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

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

秀丽线虫纵向成像的微流控平台

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

生物工程

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 ...

Tracking Calcium in the Early Developmental Phases of C. elegans

In Vivo Visualization of Calcium Transients during Fertilization and Early Development in C. elegans

Calcium is an important signaling molecule during the oocyte-to-embryo transition (OET) and early embryogenesis. The hermaphroditic nematode Caenorhabditis elegans provides several unique advantages for the study of the OET as it is transparent and has an ordered gonad that produces one mature oocyte every ~23 min at 20 &#176;C. We have modified the genetically encoded calcium indicator jGCaMP7s to fluorescently indicate the moment of fertilization within a living organism. We have termed this reporter &#34;CaFE&#34; for Calcium during Fertilization in C. elegans. The CaFE reporter was&#160;engineered into a safe harbor locus in single copy, has no significant impact on physiology or fecundity, and produces a robust signal upon fertilization. Here, a series of protocols is presented for utilizing the CaFE reporter as an in vivo tool for dissecting the OET and embryogenesis. We include methods to synchronize worms, examine the effects of RNAi knockdown, mount worms for imaging, and to visualize calcium in oocytes and embryos. Additionally, we present the generation of additional worm strains to aid in this type of analysis. Demonstrating the utility of the CaFE reporter to visualize the timing of fertilization, we report that double ovulation occurs when ipp-5 is targeted by RNAi and that only the first oocyte undergoes immediate fertilization. Furthermore, the discovery of single-cell calcium transients during early embryogenesis is reported here, demonstrating that the CaFE reporter persists into early development. Importantly, the CaFE reporter in worms is simple enough to use for incorporation into course-based undergraduate research (CURE) laboratory classes. The CaFE reporter, coupled with the ordered gonad and ease of RNAi in worms, facilitates inquiry into the cell-cell dynamics required to regulate internal fertilization and early embryogenesis.

Here, we present protocols and tools for visualizing calcium during fertilization and early embryogenesis using a genetically encoded calcium reporter expressed in the germline of the model nematode C. elegans.

Calcium is an important signaling molecule during the oocyte-to-embryo transition (OET) and early embryogenesis. The hermaphroditic nematode ...

发育生物学

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.
Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 &#181;m and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. &#8195;

比率钙显像对单个神经元的行为线虫线虫

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...

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 ...

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a crucial component of intracellular signaling. The visualization of subcellular Ca2+ dynamics in vivo,&#160;where neurons can be studied in their native, intact circuitry, has proven technically challenging in complex nervous systems. The transparency and relatively simple nervous system of the nematode Caenorhabditis elegans enable the cell-specific expression and in vivo visualization of fluorescent tags and indicators. Among these are fluorescent indicators that have been modified for use in the cytoplasm as well as various subcellular compartments, such as the mitochondria. This protocol enables non-ratiometric Ca2+ imaging in vivo with a subcellular resolution that permits the analysis of Ca2+ dynamics down to the level of individual dendritic spines and mitochondria. Here, two available genetically encoded indicators with different Ca2+ affinities are used to demonstrate the use of this protocol for measuring relative Ca2+ levels within the cytoplasm or mitochondrial matrix in a single pair of excitatory interneurons (AVA). Together with the genetic manipulations and longitudinal observations possible in C. elegans, this imaging protocol may be useful for answering questions regarding how Ca2+ handling regulates neuronal function and plasticity.

Subcellular Imaging of Neuronal Calcium Handling In Vivo

The current methods describe a non-ratiometric approach for high-resolution, sub-compartmental calcium imaging in vivo in Caenorhabditis elegans using readily available genetically encoded calcium indicators.

体内神经元钙处理的亚细胞成像

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a ...

Calcium Imaging: A Method to Visualize Neural Activity in Live C. elegans

Source: Turek, M. et al. <a href="https://www.jove.com/video/52742/agarose-microchambers-for-long-term-calcium-imaging-caenorhabditis">Agarose Microchambers for Long-term Calcium Imaging of&#160;Caenorhabditis elegans.</a>&#160;J. Vis. Exp.&#160;(2015).
This video introduces the concepts behind calcium imaging and includes an example protocol for time-lapse imaging of worms contained in molded agarose wells.

钙成像:一种可视化活秀丽隐杆线虫神经活动的方法

Source: Turek, M. et al. Agarose Microchambers for Long-term Calcium Imaging of&#160;Caenorhabditis elegans.&#160;J. Vis. Exp.&#160;(2015).
This video ...

琼脂糖微室的长期钙成像

摘要

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此视频中的章节

在 Vivo 钙成像中，在 C. elegans身体壁肌肉

C.在体内神经元钙成像技术线虫

自由行为秀丽隐杆线虫的钙成像，具有良好控制，非局部化振动

秀丽线虫纵向成像的微流控平台

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

In Vivo Visualization of Calcium Transients during Fertilization and Early Development in C. elegans

比率钙显像对单个神经元的行为线虫线虫

用于秀丽隐杆线虫长期生长和成像的简单微流控芯片

体内神经元钙处理的亚细胞成像

钙成像:一种可视化活秀丽隐杆线虫神经活动的方法

琼脂糖微室的长期钙成像

摘要

探索更多视频

此视频中的章节

在 Vivo 钙成像中，在 C. elegans身体壁肌肉

C.在体内神经元钙成像技术线虫

自由行为 秀丽隐杆线虫 的钙成像，具有良好控制，非局部化振动

秀丽线虫纵向成像的微流控平台

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

In Vivo Visualization of Calcium Transients during Fertilization and Early Development in C. elegans

比率钙显像对单个神经元的行为线虫线虫

用于秀丽隐杆线虫长期生长和成像的简单微流控芯片

体内神经元钙处理的亚细胞成像

钙成像:一种可视化活秀丽隐杆线虫神经活动的方法

自由行为秀丽隐杆线虫的钙成像，具有良好控制，非局部化振动