A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors.
A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors.
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结构化照明的指南TIRF显微镜，高速带有多种颜色。
DOI：
10.3791/53988
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发表时间：
2016-05-30
期刊：
影响因子：
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通讯作者：
Kaminski CF
中科院分区：
文献类型：
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作者：
Young LJ;Ströhl F;Kaminski CF
结构照明显微镜（SIM）光学超分辨成像是化学和生物医学科学中分子水平过程可视化的关键技术。虽然商业SIM系统是可用的，但在实验室中定制设计的系统可以优于商业系统，后者通常被设计用于易于使用和通用应用，无论是在成像保真度还是速度方面。本文介绍了如何构建SIM系统的深入指南，该系统使用全内反射（TIR）照明，能够以高达10 Hz的频率以三种颜色成像，分辨率达到100 nm。由于SIM和TIRF的结合，该系统提供了比竞争对手技术更好的图像对比度。为了实现这些规格，使用若干光学元件来实现对所有可用激发波长的照明光的偏振状态和空间结构的自动控制。给出了硬件实现和控制的全部细节，以实现激发光图案生成、波长、偏振状态和相机控制之间的同步，重点是实现最大采集帧速率。提出了一种逐步的系统对准和校准方案，并在理想的测试样品上验证了可实现的分辨率提高。用活细胞证明了视频速率超分辨率成像的能力。
Optical super-resolution imaging with structured illumination microscopy (SIM) is a key technology for the visualization of processes at the molecular level in the chemical and biomedical sciences. Although commercial SIM systems are available, systems that are custom designed in the laboratory can outperform commercial systems, the latter typically designed for ease of use and general purpose applications, both in terms of imaging fidelity and speed. This article presents an in-depth guide to building a SIM system that uses total internal reflection (TIR) illumination and is capable of imaging at up to 10 Hz in three colors at a resolution reaching 100 nm. Due to the combination of SIM and TIRF, the system provides better image contrast than rival technologies. To achieve these specifications, several optical elements are used to enable automated control over the polarization state and spatial structure of the illumination light for all available excitation wavelengths. Full details on hardware implementation and control are given to achieve synchronization between excitation light pattern generation, wavelength, polarization state, and camera control with an emphasis on achieving maximum acquisition frame rate. A step-by-step protocol for system alignment and calibration is presented and the achievable resolution improvement is validated on ideal test samples. The capability for video-rate super-resolution imaging is demonstrated with living cells.