简单的微流控芯片的<em在体内</em>成像<em> C.线虫</em><em>果蝇</em>和斑马鱼
Summary
一个简单的微流体装置已经发展到进行麻醉免费<em在体内</em>成像<em> C.线虫</em>完整<em>果蝇</em幼虫和斑马鱼幼虫。该设备采用可变形的PDMS膜固定的模式生物，以执行时间的推移成像的许多过程，如心脏的跳动，细胞分裂和亚细胞神经传输。我们证明了使用本设备，并从不同的模型系统收集的数据的不同类型的显示例子。
Abstract
微机电制流体设备提供一个可访问的微环境中的小型生物体内研究。简单的制造工艺可用于微流体装置使用软光刻技术1-3。亚细胞成像4,5，微流体器件已被用于在体内激光显微2,6和细胞成像4,7 在体内成像需要固定的生物体。取得这样的成绩使用吸力5,8，锥形通道6,7,9，变形膜2-4,10，吸气额外的冷却5，麻醉气体，温度敏感凝胶12，氰基丙烯酸酯胶13和麻醉剂如左旋咪唑14 ，15。常用麻醉药影响突触传递16,17和是已知的亚细胞神经传输4上有不利影响。在这种ST乌德我们展示了一个基于聚二甲基硅氧烷（PDMS）的装置，可以让完整的遗传模式生物，如线虫线虫 ， 果蝇和斑马鱼幼虫的麻醉剂无固定膜。这些模式生物在体内研究微流体装置，因为他们小的直径和光学透明或半透明的机构适合。体直径范围从〜10微米〜800微米的早期幼虫阶段C.线虫和斑马鱼幼虫和要求的不同尺寸的微流体装置，实现完整的固定的高分辨率时间的推移成像。这些生物体被固定使用施加的压力由压缩氮气通过液体柱和成像使用倒置显微镜。从陷阱释放的动物在10分钟之内返回到正常运动。
我们演示了四个应用程序时移成像C.线虫即，成像线粒体运输中的神经元，突触前囊泡运输中的运输缺陷的突变体，谷氨酸受体运输和Q神经母细胞分裂。从这样的电影中获得的数据，表明微流的固定化是一个有用的和准确的方法，麻醉动物（ 图1J和3C-F 4）相比时， 在体内细胞和亚细胞事件的数据的获取。
设备外形尺寸进行修改，以允许时间推移成像的不同阶段C.的一龄果蝇和斑马鱼幼虫。运输标记GFP（syt.eGFP）在感觉神经元的突触小泡的基本完整的一龄果蝇幼虫表达的胆碱能神经的感觉神经元中的突触囊泡的标记显示定向运动。一个类似的装置已经被用来进行时间推移成像的心跳在受精后30小时（HPF）斑马鱼幼虫。这些数据表明，我们已经开发的简单的设备可以适用于各种各样的模型系统，以研究几个细胞在体内的生物和发育的现象。
Protocol
1。 SU8主制造设计的微流体的结构使用Clewin软件和打印它使用65,024 DPI激光绘图仪与最小特征尺寸为8μm的对电路板膜。 清洁2厘米×2厘米的硅晶片，与在20％的KOH，持续1分钟，并在去离子水中冲洗，每一个晶片的流动层和其相应的控制层的原生氧化层。 件用氮气吹干，4小时，脱水，在120℃的热板上。允许的作品冷却至室温，然后再进行下一个步骤。 将硅块在时间上的?…
Representative Results
固定装置是一个通过粘接两层制成的双层PDMS块：的流动层（第1层）和一个控制层（第2层），如在图1中所示。的主要的陷阱被连接到氮气瓶通过一个调节器和一个三路停止旋塞应用必要的（3-14磅）的压力通过液体柱（ 图1A）到膜上。偏转膜固定C。线虫 ， 果蝇或斑马鱼幼虫在具有不同尺寸（ 图1B和1C）的流动通道设计。 C.不?…
Discussion
PDMS微流体装置是光学透明的，因此，可用于任何透明/半透明的模型生物体在体内成像的高分辨率。我们的设计是适合高倍率完整的活的动物细胞和亚细胞事件的时空成像。微细加工用软光刻技术，可轻松操作的模式生物的各种尺寸的设备尺寸。 C.不同阶段制造各种尺寸的移动设备线虫 ， 果蝇和斑马鱼幼虫。移动设备具有不同的高度为40μm和80μm，在其流层通道?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
我们感谢果蝇股，Tarjani阿加瓦尔博士Krishanu雷为保持果蝇笼，彼得·若为nuIs25和CGC为C.的菌株。 SPK取得jsIs609在迈克尔·九重实验室。我们感谢他的帮助下，在微流体装置jsIs609动物的线粒体运输时间的推移成像Arpan Agnihotri（BITS Pilani的）。我们非常感谢博士瓦沙拉Thirumalai和Surya普拉卡什为我们提供了与斑马鱼的胚胎。我们感谢博士奎师那和CIFF NCBS支持部科学和技术中心的纳米技术（第SR/55/NM-36-2005）的旋转盘共聚焦显微镜的使用。我们也感谢Kaustubh劳，V. Venkatraman和雀塔那Sachidanand的进行讨论。这项工作是由DBT博士后研究（SM），DST快车道计划（SM）和DBT授予（SPK）。 SA的支持由DST和CSIR SPK补助金。
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
| Silicon wafers | University wafer | 150 mm (100) Mech Grade SSP Si | |
| Clewin Software | WieWeb software | Version 2.90 | |
| Laser plotter | Fine Line Imaging | 65,024 DPI | |
| HMDS | Sigma-Aldrich | 440191-100ML | |
| SU8 | Microchem | SU8-2025, SU8-2050 | |
| Developer | Microchem | SU8 Developer | |
| Silane | Sigma-Aldrich | 448931-10G | |
| PDMS | Dow corning | Sylgard 184 | |
| UV lamp | Oriel | 66943 | 200W Hg Oriel Light |
| Hot air oven | Ultra Instruments | Custom made | Set at 50 °C |
| Hot plate | IKA Laboratory Equipment | 3810000 | http://www.ika.com |
| Plasma cleaner | Harrick Plasma | PDC-32G | |
| Spinner | Semiconductor Production Systems | SPIN150-NPP | www.SPS-Europe.com |
| Glass cover slip | Gold Seal | 22 X 22 mm, No. 1 thickness | |
| C. elegans | Caenorhabditis Genetics Center (CGC) | e1265, ayIs4 | |
| Drosophila | Bloomington | P{chaGAL4}/cyo, UAS-syt.eGFP | |
| Zebrafish | Indian wild type | Wild type | |
| Tygon tube | Sigma | Z279803 | |
| Micro needle | Sigma | Z118044 | Cut into 1 cm pieces |
| 3-way stopcock | Sigma | S7521 | |
| Harris puncher | Sigma | Z708631 | |
| Compressed nitrogen gas | Local Gas supplier | Use a regulator to control the pressure | |
| Stereo microscope | Nikon | SMZ645 | |
| Confocal microscope | Andor & Olympus | Yokogawa spinning disc confocal microscope | |
| ImageJ | National Institutes of Health | www.rsbweb.nih.gov/ij | Java based image processing program |
Referenzen
- Whitesides, G. M., Ostuni, E., Takayama, S., Jiang, X., Ingber, D. E. Soft lithography in biology and biochemistry. Annu. Rev. Biomed. Eng. 3, 335-373 (2001).
- Guo, S. X. Femtosecond laser nanoaxotomy lab-on-a-chip for in vivo nerve regeneration studies. Nat. Methods. 5, 531-533 (2008).
- Gilleland, C. L., Rohde, C. B., Zeng, F., Yanik, M. F. Microfluidic immobilization of physiologically active Caenorhabditis elegans. Nat. Protoc. 5, 1888-1902 (2010).
- Mondal, S., Ahlawat, S., Rau, K., Venkataraman, V., Koushika, S. P. Imaging in vivo neuronal transport in genetic model organisms using microfluidic devices. Traffic. 12, 372-385 (2011).
- Chung, K., Crane, M. M., Lu, H. Automated on-chip rapid microscopy, phenotyping and sorting of C. elegans. Nat. Methods. 5, 637-643 (2008).
- Allen, P. B. Single-synapse ablation and long-term imaging in live C. elegans. J. Neurosci. Methods. 173, 20-26 (2008).
- Chronis, N., Zimmer, M., Bargmann, C. I. Microfluidics for in vivo imaging of neuronal and behavioral activity in Caenorhabditis elegans. Nat. Methods. 4, 727-731 (2007).
- Rohde, C. B., Zeng, F., Gonzalez-Rubio, R., Angel, M., Yanik, M. F. Microfluidic system for on-chip high-throughput whole-animal sorting and screening at subcellular resolution. Proc. Natl. Acad. Sci. U.S.A. 104, 13891-13895 (2007).
- Hulme, S. E., Shevkoplyas, S. S., Apfeld, J., Fontana, W., Whitesides, G. M. A microfabricated array of clamps for immobilizing and imaging C. elegans. Lab Chip. 7, 1515-1523 (2007).
- Zeng, F., Rohde, C. B., Yanik, M. F. Sub-cellular precision on-chip small-animal immobilization, multi-photon imaging and femtosecond-laser manipulation. Lab Chip. 8, 653-656 (2008).
- Chokshi, T. V., Ben-Yakar, A., Chronis, N. CO2 and compressive immobilization of C. elegans on-chip. Lab Chip. 9, 151-157 (2009).
- 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).
- Goodman, M. B., Hall, D. H., Avery, L., Lockery, S. R. Active currents regulate sensitivity and dynamic range in C. elegans neurons. Neuron. 20, 763-772 (1998).
- Snow, J. J. Two anterograde intraflagellar transport motors cooperate to build sensory cilia on C. elegans neurons. Nat. Cell Biol. 6, 1109-1113 (2004).
- Lewis, J. A., Wu, C. H., Berg, H., Levine, J. H. The genetics of levamisole resistance in the nematode Caenorhabditis elegans. Genetik. 95, 905-928 (1980).
- Richmond, J. E., Jorgensen, E. M. One GABA and two acetylcholine receptors function at the C. elegans neuromuscular junction. Nat. Neurosci. 2, 791-797 (1999).
- Badre, N. H., Martin, M. E., Cooper, R. L. The physiological and behavioral effects of carbon dioxide on Drosophila melanogaster larvae. Comp. Biochem. Physiol. A. Mol. Integr. Physiol. 140, 363-376 (2005).
- Stiernagle, T. Maintenance of C. elegans. WormBook. , 1-11 (2006).
- Westerfield, M. The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). , (2000).
- Henn, K., Braunbeck, T. Dechorionation as a tool to improve the fish embryo toxicity test (FET) with the zebrafish (Danio rerio). Comp. Biochem. Physiol. C. Toxicol. Pharmacol. 153, 91-98 (2011).
- Fatouros, C. Inhibition of tau aggregation in a novel Caenorhabditis elegans model of tauopathy mitigates proteotoxicity. Hum. Mol. Genet. , (2012).
- Barkus, R. V., Klyachko, O., Horiuchi, D., Dickson, B. J., Saxton, W. M. Identification of an axonal kinesin-3 motor for fast anterograde vesicle transport that facilitates retrograde transport of neuropeptides. Mol. Biol. Cell. 19, 274-283 (2008).
- Craig, M. P., Gilday, S. D., Hove, J. R. Dose-dependent effects of chemical immobilization on the heart rate of embryonic zebrafish. Lab. Anim. (NY). 35, 41-47 (2006).
- Pardo-Martin, C. High-throughput in vivo vertebrate screening. Nat. Methods. 7, 634-636 (2010).
- Stainier, D. Y., Lee, R. K., Fishman, M. C. Cardiovascular development in the zebrafish. I. Myocardial fate map and heart tube formation. Development. 119, 31-40 (1993).
- Hall, D. H., Hedgecock, E. M. Kinesin-related gene unc-104 is required for axonal transport of synaptic vesicles in C. elegans. Cell. 65, 837-847 (1991).
- Kumar, J. The Caenorhabditis elegans Kinesin-3 motor UNC-104/KIF1A is degraded upon loss of specific binding to cargo. PLoS Genet. 6, e1001200 (2010).
- Ou, G., Vale, R. D. Molecular signatures of cell migration in C. elegans Q neuroblasts. J. Cell. Biol. 185, 77-85 (2009).
- Levitan, E. S., Lanni, F., Shakiryanova, D. In vivo imaging of vesicle motion and release at the Drosophila neuromuscular junction. Nat. Protoc. 2, 1117-1125 (2007).
- Morris, R. L., Hollenbeck, P. J. The regulation of bidirectional mitochondrial transport is coordinated with axonal outgrowth. J. Cell. Sci. 104, 917-927 (1993).
- Louie, K., Russo, G. J., Salkoff, D. B., Wellington, A., Zinsmaier, K. E. Effects of imaging conditions on mitochondrial transport and length in larval motor axons of Drosophila. Comp. Biochem. Physiol. A. Mol. Integr. Physiol. 151, 159-172 (2008).
- Pilling, A. D., Horiuchi, D., Lively, C. M., Saxton, W. M. Kinesin-1 and Dynein are the primary motors for fast transport of mitochondria in Drosophila motor axons. Mol. Biol. Cell. 17, 2057-2068 (2006).
Diesen Artikel zitieren
Mondal, S., Ahlawat, S., Koushika, S. P. Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish. J. Vis. Exp. (67), e3780, doi:10.3791/3780 (2012).