Park Seongjin, Zhang Jiacheng, Reyer Matthew A, Zareba Joanna, Troy Andrew A, Fei Jingyi
Department of Biochemistry and Molecular Biology, University of Chicago.
The Institute for Biophysical Dynamics, University of Chicago.
J Vis Exp. 2018 Oct 28(140):58320. doi: 10.3791/58320.
Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to conventional epi-fluorescence microscopy, various imaging techniques have been developed to achieve specific experimental goals. Some of the widely used techniques include single-molecule fluorescence resonance energy transfer (smFRET), which can report conformational changes and molecular interactions with angstrom resolution, and single-molecule detection-based super-resolution (SR) imaging, which can enhance the spatial resolution approximately ten to twentyfold compared to diffraction-limited microscopy. Here we present a customer-designed integrated system, which merges multiple imaging methods in one microscope, including conventional epi-fluorescent imaging, single-molecule detection-based SR imaging, and multi-color single-molecule detection, including smFRET imaging. Different imaging methods can be achieved easily and reproducibly by switching optical elements. This set-up is easy to adopt by any research laboratory in biological sciences with a need for routine and diverse imaging experiments at a reduced cost and space relative to building separate microscopes for individual purposes.
荧光显微镜是一种强大的工具，可用于原位检测生物分子，并实时监测其动态和相互作用。除了传统的落射荧光显微镜外，还开发了各种成像技术以实现特定的实验目标。一些广泛使用的技术包括单分子荧光共振能量转移（smFRET），它可以以埃分辨率报告构象变化和分子相互作用；以及基于单分子检测的超分辨率（SR）成像，与衍射极限显微镜相比，它可以将空间分辨率提高约十到二十倍。在这里，我们展示了一个客户定制的集成系统，该系统在一台显微镜中融合了多种成像方法，包括传统的落射荧光成像、基于单分子检测的SR成像以及多色单分子检测，包括smFRET成像。通过切换光学元件，可以轻松且可重复地实现不同的成像方法。相对于为单个目的建造单独的显微镜，这种设置对于任何需要进行常规和多样成像实验的生物科学研究实验室来说都很容易采用，且成本和空间更低。
