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
Summary
美国匹茨堡研究的新工具需要了解机械应力是如何激活生化通路并引起生物学反应的。在这里, 我们展示了一种新的方法, 选择性机械刺激固定化动物与微流控器陷阱, 允许高分辨率成像的细胞反应。
Abstract
美国匹茨堡的一个中心目标是了解机械应力对蛋白质和细胞的相互作用。尽管它的重要性, 机械应力对细胞功能的影响仍然是不太清楚。在某种程度上, 这种知识差距之所以存在, 是因为很少有工具能使组织和细胞同时变形, 活动物的细胞活动成像, 以及在其他高度流动的模型生物体中有效限制运动, 如线虫秀丽线虫。小的C. 线虫使它们与基于微流体的研究设备非常匹配, 并利用微流控装置提出了固定化解决方案。虽然这些设备允许高分辨率成像, 该动物是完全包裹在烷和玻璃, 限制物理访问提供机械力或电生理记录。最近, 我们创建了一个设备, 集成气动执行器与陷阱设计兼容高分辨率荧光显微镜。所提出的驱动通道是由一个薄的, 由一个小的反式的双膜片分离的蠕虫捕获通道。这种隔膜通过施加外部源的压力偏转到蠕虫的一侧。该装置可以针对单个 mechanosensitive 神经元。这些神经元的活化是用基因编码的钙指标进行高分辨率成像的。本文介绍了在触摸受体神经元 (TRNs) 中, 使用线虫菌株表达钙敏感活性指标 (GCaMP6s) 的一般方法。然而, 该方法并不局限于 TRNs 或钙传感器作为探针, 但可以扩展到其他机械敏感的细胞或传感器。
Introduction
触觉为动物提供了关于环境的关键信息。根据施加的力量, 触摸被认为是无害的, 愉悦的, 或痛苦的。在接触过程中的组织变形是由嵌入在皮肤中的专门 mechanoreceptor 细胞检测到的, 表达受体蛋白, 最常见的离子通道。在接触和疼痛过程中, 连接力知觉与离子通道活化的步骤没有完全理解。对于皮肤组织如何过滤机械变形以及 mechanoreceptors 检测应变或应力变化的情况 (如1、2、3), 更甚了解。这种理解上的差距, 部分是由于缺乏适当的工具, 在观察细胞水平的反应时, 对活着的动物的皮肤表面施加精确的机械刺激。虽然原子力显微镜已广泛用于应用和测量孤立细胞中的作用力4,5和激活 Piezo1 受体在活细胞6, 类似的实验使用活体动物, 特别是C. 线虫, 由于这个主题的内在流动性, 而臭名昭著的挑战性。这一挑战传统上绕过使用兽医或外科级氰胶水固定在琼脂垫上的个别动物,1,7,8,9。这种方法是有成效的, 但有限制与固定的技能所需的胶合和软琼脂表面上的机械依从性。微流体策略是一种免费的替代方法, 可以避免一些与胶合有关的并发症。
线虫线虫是一个基因模型有机体与完全映射神经系统, 由于动物的大小, 是一个很好的适合微流体技术. 微流体的设备提供的优势, 否则极端移动动物可以克制, 而执行高分辨率成像和提供相关的神经调节刺激。在微流控技术的帮助下, 活着的动物可以不受伤害地固定化10,11, 启用对整个生存期1213和高分辨率的行为活动的监视神经元活动的成像14,15,16,17。此外, 许多 mechanoreceptor 神经元需要的触觉和疼痛的特点, 可以在他们的生理1,8, 机械4,18,19, 和分子级别20,21,22。
C. 线虫用六 TRNs 对其体壁进行柔和的机械刺激, 其中三支配动物的前 (ALML/r 和 AVM), 三支配动物的后部 (PLML/r 和 PVM)。在它的 TRNs8中广泛研究了传感所需的离子通道分子在生化信号中的应用。本文介绍了一个微流控平台23 , 使研究人员能够将精确的机械力应用于固定化的C. 线虫蛔虫的皮肤, 同时通过光学成像读取其内部组织的变形。除了提供良好定义的机械刺激, 钙瞬变可以记录在 mechanoreceptor 神经元的亚细胞分辨率和相关的形态学和解剖特征。该设备由一个中央补漏白通道组成, 它包含一个动物, 并在六个气动驱动通道 (图 1和图 2) 旁边呈现其外观。六通道沿陷印通道定位, 为蠕虫的六 TRNs 提供机械刺激。这些通道通过薄的可由外部气压源 (图 1) 驱动的薄壁膜片与陷印室隔开。我们对压力的偏转进行了校准, 并提供了本文的测量结果。每个执行器可以单独解决, 并用于刺激 mechanoreceptor 的选择。压力是通过压电驱动的压力泵提供的, 但任何替代装置都可以使用。我们表明, 压力协议可以用来激活 TRNs在体内和演示的操作设备, 以提供机械刺激成人C. 线虫, 加载成年动物进入设备, 执行钙成像实验, 分析结果。器件制造包括两个主要步骤: 1) 光刻, 使模具从 SU-8;2) 模压成型机制造设备。为了简洁明了, 读者可以参考以前发布的文章和协议24,25 , 了解如何生成模具和设备的说明。
Protocol
1. 设备制造 下载附加的掩码文件 (补充文件 1), 并使用商业服务或内部设施生成 chrome 掩码。由于器件的最小尺寸为10µm (执行器膜厚度), 确保掩模具有足够高的分辨率, 在0.25 µm 内, 可靠地产生特性。 遵循标准的 SU-8 光刻方法 (例如, 引用24,25,26), 以制造随后生产…
Representative Results
SU-8 光刻与芯片粘合光刻协议和注塑工艺遵循标准程序。详细信息可在其他地方找到23,24,25,26。在固化后, 应将硅片剥离, 无问题。如果 SU-8 的特性在其剥离过程中脱落, SU-8 黏附层或硅烷化是不够的。如果等离子活化的玻璃盖玻片和非晶硅芯片是不够的, 弱粘结?…
Discussion
该协议说明了一种方法, 提供精确的机械刺激的皮肤的蛔虫被困在微流控芯片。它的目的是促进物理刺激的综合回答生物学问题, 并旨在简化生物实验室的美国匹茨堡研究。这种方法扩展了以前的化验, 以评估 mechanosensory 神经元在C. 线虫中的作用。以前测量的定量和半定量技术力1、34和行为35, 但很难与神经元活动的高分辨率成像…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们感谢桑德拉 n. Manosalvas-Kjono, 普珥节 Ladpli, 法拉梅蒙, Divya Gopisetty, 和维罗妮卡支持在设备设计和产生变种动物。这项研究得到了 NIH 赠款 R01EB006745 (BLP)、R01NS092099 (MBG)、K99NS089942 (至 MK)、F31NS100318 (氮化铝) 的支持, 并获得欧洲联盟2020研究和创新计划 (协调委员会) 的资助 (授予 MK 715243 号协议)。
Materials
| Chrome mask | Compugraphics (http://www.compugraphics-photomasks.com/) | 5'', designed in AutoCAD (Autodesk, Inc.) | |
| Chrome mask | Mitani-Micronics (http://www.mitani-micro.co.jp/en/) | 5'', designed in AutoCAD (Autodesk, Inc.) | |
| Chrome mask | Kuroda-Electric (http://www.kuroda-electric.eu/ | 5'', designed in AutoCAD (Autodesk, Inc.) | |
| 4'' Silicon wafer (B-test) | Stanford Nanofabrication Facility | ||
| SU-8 2002 | MicroChem | ||
| SU-8 2050 | MicroChem | ||
| Spin-coater | Laurell Technologies | WS-400BZ-6NPP/LITE | |
| Exposure timer | Optical Associates, Inc | OAI 150 | |
| Illumination controller | Optical Associates, Inc | 2105C2 | |
| SU-8 developer | MicroChem | ||
| 2-Propanol | Fisher Scientific | A426F-1GAL | |
| Acetone | Fisher Scientific | A18-4 | |
| Trichloromethylsilane (TCMS) | Sigma-Aldrich | 92361-500ML | Caution: TCMS is toxic and water-reactive |
| Sylgard 184 Elastomer Kit | Dow Corning | PDMS prepolymer | |
| Biopsy punch, 1 mm | VWR | 95039-090 | |
| Oxygen Plasma Asher | Branson/IPC | ||
| Small metal tubing (0.635 mm OD, 0.4318 mm ID, 12.7 mm long); gage size 23TW | New England Small Tube Corporation | NE-1300-01 | |
| Nalgene syringe filter, 0.22 μm | Thermo Scientific | 725-2520 | to filter all solution, small particles would clog the chip |
| Polyethylene tubing; 0.9652 mm OD, 0.5842 mm ID | Solomon Scientific | BPE-T50 | |
| Syringe, 1 ml | BD Scientific | 309628 | for worm trapping and release |
| Syringe, 20 ml | BD Scientific | 309661 | for gravity-based flow |
| Gilson Minipuls 3, Peristaltic pump | Gilson | to suck solutions and worms out of the chip | |
| Microfluidic flow controller, equipped with 0–800 kPa pressure channel | Elveflow | OB1 MK3 | pressure delivery |
| Water-Resistant Clear Poly- urethane Tubing, 4 mm ID and 6 mm OD | McMaster-Carr | 5195 T52 | connection from house air to pressure pump |
| Water-Resistant Clear Polyurethane Tubing, 2.6mm ID and 4mm OD | McMaster-Carr | 5195 T51 | connect pressure pump to small tubng |
| Push-to-Connect Tube Fitting for Air | McMaster-Carr | 5111K468 | metric – imperial converter |
| Straight Connector for 6 mm × 1/4″ Tube OD | McMaster-Carr | 5779 K258 | |
| Leica DMI 4000 B microscopy system | Leica | ||
| 63×/1.32 NA HCX PL APO oil objective | Leica | 506081 | |
| Hamamatsu Orca-Flash 4.0LT digital CMOS camera | Hamamatsu | C11440-42U | |
| Lumencor Spectra X light engine | Lumencor | With cyan and green/yellow light source | |
| Excitation beam splitter | Chroma | 59022bs | in the microscope |
| Hamamatsu W-view Gemini Image splitting optics | Hamamatsu | A12801-01 | to split green and red emission and project them on different areas on the camera chip |
| Emission beam splitter | Chroma | T570lpxr | in the image splitter |
| Emission filters GCamp6s | Chroma | ET525/50m | in the image splitter |
| Emission filters mCherry | Chroma | ET632/60m | in the image splitter |
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Citer Cet Article
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