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首页> 美国卫生研究院文献>Journal of Visualized Experiments : JoVE >Visualization of Cortex Organization and Dynamics in Microorganisms using Total Internal Reflection Fluorescence Microscopy
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Visualization of Cortex Organization and Dynamics in Microorganisms using Total Internal Reflection Fluorescence Microscopy

机译：使用全内反射荧光显微镜观察皮层组织和微生物动力学

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TIRF microscopy has emerged as a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules in vitro and in living cells1. The optical phenomenon of total internal reflection occurs when light passes from a medium with high refractive index into a medium with low refractive index at an angle larger than a characteristic critical angle (i.e. closer to being parallel with the boundary). Although all light is reflected back under such conditions, an evanescent wave is created that propagates across and along the boundary, which decays exponentially with distance, and only penetrates sample areas that are 100-200 nm near the interface2. In addition to providing superior axial resolution, the reduced excitation of out of focus fluorophores creates a very high signal to noise ratios and minimizes damaging effects of photobleaching2,3. Being a widefield technique, TIRF also allows faster image acquisition than most scanning based confocal setups.At first glance, the low penetration depth of TIRF seems to be incompatible with imaging of bacterial and fungal cells, which are often surrounded by thick cell walls. On the contrary, we have found that the cell walls of yeast and bacterial cells actually improve the usability of TIRF and increase the range of observable structures4-8. Many cellular processes can therefore be directly accessed by TIRF in small, single-cell microorganisms, which often offer powerful genetic manipulation techniques. This allows us to perform in vivo biochemistry experiments, where kinetics of protein interactions and activities can be directly assessed in living cells.We describe here the individual steps required to obtain high quality TIRF images for Saccharomyces cerevisiae or Bacillus subtilis cells. We point out various problems that can affect TIRF visualization of fluorescent probes in cells and illustrate the procedure with several application examples. Finally, we demonstrate how TIRF images can be further improved using established image restoration techniques.
机译：TIRF显微镜已经成为一种强大的成像技术，可以研究荧光分子在体外和在活细胞中的时空动态 1 。当光以大于特征临界角的角度（即更接近于平行于边界）从具有高折射率的介质进入具有低折射率的介质时，发生全内反射的光学现象。尽管在这种情况下所有光都被反射回去，但会产生一个wave逝波，该e逝波沿边界传播并沿边界传播，并随距离呈指数衰减，并且仅穿透界面 2 。除了提供出色的轴向分辨率外，聚焦荧光团激发的减少也产生了很高的信噪比，并使光漂白 2,3 的破坏作用最小化。作为一种广域技术，TIRF还比大多数基于扫描的共聚焦设置提供了更快的图像获取。乍一看，TIRF的低穿透深度似乎与细菌和真菌细胞的成像不兼容，细菌和真菌细胞通常被厚厚的细胞壁所包围。相反，我们发现酵母和细菌细胞的细胞壁实际上提高了TIRF的可用性，并增加了可观察结构的范围 4-8 。因此，TIRF可以直接在小型单细胞微生物中访问许多细胞过程，这些微生物通常提供强大的遗传操作技术。这使我们能够进行体内生物化学实验，在其中可以直接在活细胞中评估蛋白质相互作用和活性的动力学。在此，我们描述了获得啤酒酵母或枯草芽孢杆菌细胞高质量TIRF图像所需的各个步骤。我们指出了可能影响细胞中荧光探针的TIRF可视化的各种问题，并通过几个应用示例说明了该过程。最后，我们演示了如何使用已建立的图像恢复技术进一步改善TIRF图像。

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