构建具有主动稳定功能（反馈式单分子定位显微镜）的全内反射显微镜（TIRF）。
Building a Total Internal Reflection Microscope (TIRF) with Active Stabilization (Feedback SMLM).
作者信息
Coelho Simao, Baek Jongho, Gooding J Justin, Gaus Katharina
机构信息
EMBL Australia Node in Single Molecule Science, School of Medical Sciences, University of New South Wales, Sydney, Australia.
ARC Centre of Excellence in Advanced Molecular Imaging, University of New South Wales, Sydney, Australia.
出版信息
Bio Protoc. 2021 Jul 5;11(13):e4074. doi: 10.21769/BioProtoc.4074.
The data quality of high-resolution imaging can be markedly improved with active stabilization, which is based on feedback loops within the microscope that maintain the sample in the same location throughout the experiment. The purpose is to provide a highly accurate focus lock, therefore eliminating drift and improving localization precision. Here, we describe a step-by-step protocol for building a total internal reflection microscope combined with the feedback loops necessary for sample and detection stabilization, which we routinely use in single-molecule localization microscopy (SMLM). The performance of the final microscope with feedback loops, called feedback SMLM, has previously been described. We demonstrate how to build a replica of our system and include a list of the necessary optical components, tips, and an alignment strategy.
通过主动稳定技术可以显著提高高分辨率成像的数据质量，该技术基于显微镜内的反馈回路，在整个实验过程中将样品保持在同一位置。目的是提供高度精确的聚焦锁定，从而消除漂移并提高定位精度。在此，我们描述了一个逐步的方案，用于构建一台结合了样品和检测稳定所需反馈回路的全内反射显微镜，我们在单分子定位显微镜（SMLM）中经常使用该显微镜。先前已经描述了带有反馈回路的最终显微镜（称为反馈SMLM）的性能。我们展示了如何构建我们系统的复制品，并列出了必要的光学组件、技巧和对准策略。
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