利用先进的纺额圆盘全内反射荧光显微镜观察细胞粘附形成
Summary
将提供一种先进的显微镜, 可对这两种情况、隔离的质膜和周围的细胞内体积进行快速和高分辨率的成像。将纺盘和总内部反射荧光显微镜集成到一个装置中, 可以在每个图像堆栈的高采集速率下进行实时成像实验。
Abstract
在活细胞中, 粘附形成等过程涉及质膜和细胞内部的广泛结构变化。为了可视化这些高度动态的事件, 结合了两种互补的光显微镜技术, 可以快速成像实时样品: 旋转盘显微镜 (sd), 用于快速和高分辨率的体积记录和全面的内部反射荧光 (tirf) 显微镜, 用于精确定位和可视化质膜。将展示一个全面、完整的成像方案, 用于指导样品制备、显微镜校准、图像形成和采集, 从而形成具有高时空分辨率的多色 sd-tirf 实时成像系列。生成多维实时成像数据集所需的所有图像后处理步骤, 即各个通道的注册和组合,都在开源软件 imagej 的自写宏中提供。在粘附配合物的萌发和成熟过程中, 荧光蛋白的成像, 以及肌动蛋白细胞骨架网络的形成, 被用作这一新方法的原理证明。高分辨率三维显微镜和 tirf 的结合提供了对细胞环境中这些复杂过程的详细描述, 同时, 精确定位了与膜相关的分子。信噪比。
Introduction
我们的时代, 光显微镜技术提供了高/超分辨率成像的固定和活的标本是迅速发展。超分辨率技术, 如受激发射耗尽 (sted)、结构化照明显微镜 (sim) 和光激活定位显微镜 (palm) 或直接随机光学重建显微镜 (storm), 分别是商业上可获得的, 并使成像的亚细胞结构显示的细节几乎在分子规模 1,2,3, 4, 5,6。然而, 这些方法在现场成像实验中的适用性仍然有限, 在现场成像实验中, 需要以每秒多个帧的采集速度对大容量进行可视化。通过质膜调节的高度动态过程的品种,如内/外渗、粘附、迁移或信号, 在大细胞内高速发生。最近, 为了填补这一空白, 提出了一种集成的显微镜技术, 称为旋转磁盘 tirf (sd-tirf)7。具体而言, tirf 显微镜允许专门分离和定位质膜8,9, 而 sd 显微镜是最敏感和快速的实时成像技术之一, 用于可视化和跟踪细胞质中的亚细胞细胞器10,11。在过去的 12,13, 这两种成像技术的组合已经实现, 然而, 这里介绍的显微镜 (图 1) 终于符合执行实时成像 sd-tirf 实验的标准以每秒3帧的速度完成上述过程。由于这种显微镜是商业上可用的, 这份手稿的目标是详细描述, 并提供开源工具和协议的图像采集, 注册, 和可视化与 sd-tirf 显微镜。
该设置基于通过独立端口连接到两个扫描单元的倒置显微镜–左端口连接到 sd 单元, 后端口连接到 tirf 和光激活漂白实验的扫描仪单元。可用于激发的激光器 (40\ 445/488/48\ 56n–)。对于荧光信号的激发和检测, 分别采用了 100 xn1.45 油或60xéna1.49 油 tirf 目标。发射的光由双色反射镜 (561 纳米长通或561纳米长通) 分割, 并由放置在两个 em-ccd 凸轮前的各种带通滤波器 (55 纳米宽, 中心在561纳米, 54 纳米宽分别以609纳米为中心, 用于绿色和红色荧光)时代。请注意, zobiak 等人列出了有关设置的更多技术细节.7. 在 tirf 配置中, sd 单元在大约0.5 秒内移出光路, 以便可以使用相同的两个摄像机进行检测, 从而与过去13报告的大约1秒相比, 可以在两种成像模式之间更快地切换./c2 >。此功能可实现双通道同步采集, 因此可以以以前无与伦比的速度和精度执行4个通道 sd-tirf 成像。此外, sd 和 tirf 图像之间的对齐是不必要的。但是, 在开始实验之前, 必须检查两个摄像机之间的图像对齐情况, 并在必要时进行更正。在下面的协议中, 在自写 imagej 宏中实现了注册更正例程。此外, 该宏的主要目的是允许同时显示 sd 和 tirf 数据集, 尽管它们的维度不同。采集软件本身并没有提供这些功能。
Protocol
Representative Results
Discussion
本文首次成功地实现了 sd 和 tirf 显微镜, 其配置适合进行活细胞成像实验,即3个不同阶段每分钟 2个 sd-tirf 图像堆栈的高采集率。位置, 相当于总共168帧 (约3帧/秒), 被收购。前面描述的少数 sd-tirf 显微镜, 主要是缺乏足够高的成像速度, 无法在3d 中跟踪细胞过程, 在3d 中, 每个图像堆栈的时间分辨率通常小于 2 s。该装置可实现高达0.78 图像堆栈每秒, …
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们非常感谢大学医学中心汉堡-埃彭多夫的科学界为我们提供样本评估。也就是说, 我们感谢 sabine windhorst 的 NIH3T3 细胞, 感谢 andrea mordhorst 的 yfp-vinculin 和 rfp-liffolact 的马伦·鲁道夫。
Materials
| Microscope and accessories | |||
| SD-TIRF microscope | Visitron Systems | ||
| Ti with perfect focus system | Nikon | Inverted microscope stand | |
| CSU-W1 T2 | Yokogawa | Spinning disk unit in dual-camera configuration | |
| iLAS2 | Roper Scientific | TIRF/FRAP scanner | |
| Evolve | Photometrix | EM-CCD cameras | |
| PiezoZ stage | Ludl Electronic Products | Motorized Z stage | |
| Bioprecision2 XY stage | Ludl Electronic Products | Motorized XY stage | |
| Stage top incubation chamber | Okolab | Bold Line | Temperature, CO2 and humidity supply |
| Cell culture | |||
| HeLa cervical cancer cells | DSMZ | ACC-57 | |
| NIH3T3 fibroblasts | DSMZ | ACC-59 | |
| Dulbecco's phosphate buffered saline (PBS) | Gibco | 14190144 | |
| Trypsin-EDTA 0.05% | Gibco | 25300054 | |
| Dulbecco's Modified Eagle Medium + GlutaMAX-I (DMEM) | Gibco | 31966-021 | |
| OptiMEM | Gibco | 31985070 | Reduced serum medium |
| Fetal calf serum (FCS) | Gibco | 10500064 | |
| Penicillin/Streptomycin (PenStrep) | Gibco | 15140148 | |
| Full growth medium (DMEM supplemented with 10% FCS and 1% PenStrep) | |||
| TurboFect | ThermoFisher Scientific | R0531 | Transfection reagent |
| Ascorbic acid (AA) | Sigma | A544-25G | |
| 6-well cell culture plate | Sarstedt | 83.392 | |
| Glass bottom dishes | MatTek | P35G-1.5-10-C | 35mm, 0.17mm glass coverslip |
| Fibronectin, bovine plasma | ThermoFisher Scientific | 33010018 | |
| Neubauer improved chamber | VWR | 631-0696 | |
| TetraSpeck beads | ThermoFisher Scientific | T7279 | |
| Plasmids | |||
| RFP-Lifeact | Maren Rudolph, Institute of Medical Microbiology, University Medical Center Hamburg Eppendorf, Germany | ||
| YFP-Vinculin | Andrea Mordhorst, Institute of Medical Microbiology, University Medical Center Hamburg Eppendorf, Germany | ||
| Software and plugins | |||
| VisiView | Visitron Systems | Version 3 | |
| ImageJ | Version 1.52c | ||
| Turboreg plugin | http://bigwww.epfl.ch/thevenaz/turboreg/ | ||
| Macro "SD-TIRF_helper_JoVE.ijm" | this publication | https://github.com/bzobiak/ImageJ | |
| Volocity | PerkinElmer | Version 6.2.2 |
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