琼脂糖微室的长期钙成像<em>秀丽隐杆线虫</em
Summary
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.
Abstract
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 。
Introduction
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 Förster 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.
Protocol
Representative Results
Discussion
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（ http://en.wikipedia.org/wiki/List_of_microfluidics_related_companies ）。
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。
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| high-melting point agarose | Fisher Bioreagents | BP(E)164-1000 | |
| Top Vision Low Melting Point Agarose | Thermo Scientific | R0801 | |
| LED system | CoolLED | pE-2 | |
| compound microscope | Nikon | TiE | |
| stereomicroscope | Leica | M5 | |
| EMCCD camera | Andor | iXon 897 | now sold as iXon Ultra 897 |
| sCMOS camera | Andor | Neo | |
| plasma cleaner | Harrick | PDC-23G2 | |
| lid heater | MPI workshop | custom made | |
| plastic dish | Falcon | 08-757-100A | |
| z-stage for the microscope | Prior | Nano Scan Z | |
| x-y-stage for the microscope | Prior | Proscan III | |
| PDMS stamp | Abbott Laboratories | custom made | |
| red light filter | Chroma | ET660/50m | |
| 35mm petri dish | Falcon | Falcon Disposable Petri Dishes, Sterile, Corning | |
| double sided sticky tape | Sellotape/Henkel | double sided 12mm x 33 m | alternatively Advance double sided sticky tape from Rubance Adhesives can be used. |
Referencias
- Brenner, S. The genetics of Caenorhabditis elegans. Genética. 77, 71-94 (1974).
- Husson, S. J., Costa, W. S., Schmitt, C., Gottschalk, A. Keeping track of worm trackers. WormBook. , 1-17 (2012).
- Yemini, E., Jucikas, T., Grundy, L. J., Brown, A. E., Schafer, W. R. A database of Caenorhabditis elegans behavioral phenotypes. Nature Methods. 10, 877-879 (2013).
- Likitlersuang, J., Stephens, G., Palanski, K., Ryu, W. S. C. elegans tracking and behavioral measurement. Journal of Visualized Experiments : JoVE. , e4094 (2012).
- Yemini, E., Kerr, R. A., Schafer, W. R. Tracking movement behavior of multiple worms on food. Cold Spring Harbor Protocols. 2011, 1483-1487 (2011).
- Swierczek, N. A., Giles, A. C., Rankin, C. H., Kerr, R. A. High-throughput behavioral analysis in. C. elegans. Nature Methods. 8, 592-598 (2011).
- Ramot, D., Johnson, B. E., Berry, T. L., Carnell, L., Goodman, M. B. The Parallel Worm Tracker: a platform for measuring average speed and drug-induced paralysis in nematodes. PLoS One. 3, e2208 (2008).
- Larsch, J., Ventimiglia, D., Bargmann, C. I., Albrecht, D. R. High-throughput imaging of neuronal activity in Caenorhabditis elegans. Proceedings of the National Academy of Sciences U S A. 110, E4266-E4273 (2013).
- Bringmann, H. Agarose hydrogel microcompartments for imaging sleep- and wake-like behavior and nervous system development in Caenorhabditis elegans larvae. Journal of Neuroscience Methods. 201, 78-88 (2011).
- Yu, C. C., Raizen, D. M., Fang-Yen, C. Multi-well imaging of development and behavior in Caenorhabditis elegans. Journal of Neuroscience Methods. 223, 35-39 (2014).
- Luke, C. J., Niehaus, J. Z., O’Reilly, L. P., Watkins, S. C. Non-microfluidic methods for imaging live. C. elegans. Methods. 68, 542-547 (2014).
- San-Miguel, A., Lu, H. Microfluidics as a tool for C. elegans research. WormBook. , 1-19 (2013).
- Shi, W., Qin, J., Ye, N., Lin, B. Droplet-based microfluidic system for individual Caenorhabditis elegans assay. Lab Chip. 8, 1432-1435 (2008).
- Krajniak, J., Lu, H. Long-term high-resolution imaging and culture of C. elegans in chip-gel hybrid microfluidic device for developmental studies. Lab Chip. 10, 1862-1868 (2010).
- Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., Prasher, D. C. Green fluorescent protein as a marker for gene expression. Science. 263, 802-805 (1994).
- Kerr, R., et al. Optical imaging of calcium transients in neurons and pharyngeal muscle of. C. elegans. Neuron. 26, 583-594 (2000).
- Suzuki, H., et al. In vivo imaging of C. elegans mechanosensory neurons demonstrates a specific role for the MEC-4 channel in the process of gentle touch sensation. Neuron. 39, 1005-1017 (2003).
- Hilliard, M. A., et al. In vivo imaging of C. elegans ASH neurons: cellular response and adaptation to chemical repellents. EMBO Journal. 24, 63-72 (2005).
- Frokjaer-Jensen, C., et al. Effects of voltage-gated calcium channel subunit genes on calcium influx in cultured C. elegans mechanosensory neurons. Journal of Neurobiology. 66, 1125-1139 (2006).
- Kerr, R. A. Imaging the activity of neurons and muscles. WormBook. , 1-13 (2006).
- Miyawaki, A., Griesbeck, O., Heim, R., Tsien, R. Y. Dynamic and quantitative Ca2+ measurements using improved cameleons. Proceedings of the National Academy of Sciences U S A. 96, 2135-2140 (1999).
- Nakai, J., Ohkura, M., Imoto, K. A high signal-to-noise Ca(2+) probe composed of a single green fluorescent protein. Nature Biotechnology. 19, 137-141 (2001).
- Akerboom, J., et al. Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics. Frontiers in Molecular Neuroscience. 6 (2), (2013).
- Tian, L., et al. Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nature Methods. 6, 875-881 (2009).
- Edwards, S. L., et al. A novel molecular solution for ultraviolet light detection in Caenorhabditis elegans. PLoS Biology. 6, e198 (2008).
- Ward, A., Liu, J., Feng, Z., Xu, X. Z. Light-sensitive neurons and channels mediate phototaxis in C. elegans. Nat Neurosci. 11, 916-922 (2008).
- Turek, M., Lewandrowski, I., Bringmann, H. An AP2 transcription factor is required for a sleep-active neuron to induce sleep-like quiescence in. C. elegans. Current Biology. 23, 2215-2223 (2013).
- Singh, K., et al. C. elegans Notch Signaling Regulates Adult Chemosensory Response and Larval Molting Quiescence. Current Biology. 21, 825-834 (2011).
- Raizen, D. M., et al. Lethargus is a Caenorhabditis elegans sleep-like state. Nature. 451, 569-572 (2008).
- Iwanir, S., et al. The microarchitecture of C. elegans behavior during lethargus: homeostatic bout dynamics, a typical body posture, and regulation by a central neuron. Sleep. 36, 385-395 (2013).
- Nagy, S., Raizen, D. M., Biron, D. Measurements of behavioral quiescence in Caenorhabditis elegans. Methods. , (2014).
- Nagy, S., et al. A longitudinal study of Caenorhabditis elegans larvae reveals a novel locomotion switch, regulated by Galphas signaling. eLife. 2, e00782 (2013).
- Cassada, R. C., Russell, R. L. The dauerlarva, a post-embryonic developmental variant of the nematode Caenorhabditis elegans. Biología del desarrollo. 46, 326-342 (1975).
- Schwarz, J., Lewandrowski, I., Bringmann, H. Reduced activity of a sensory neuron during a sleep-like state in Caenorhabditis elegans. Current Biology. 21, R983-R984 (2011).
- Schwarz, J., Spies, J. P., Bringmann, H. Reduced muscle contraction and a relaxed posture during sleep-like Lethargus. Worm. 1, 12-14 (2012).
- Schwarz, J., Bringmann, H. Reduced sleep-like quiescence in both hyperactive and hypoactive mutants of the Galphaq Gene egl-30 during lethargus in Caenorhabditis elegans. PLoS One. 8, e75853 (2013).
