高分辨率光镊与单分子共聚焦显微镜联用
High-Resolution Optical Tweezers Combined With Single-Molecule Confocal Microscopy.
作者信息
Whitley K D, Comstock M J, Chemla Y R
机构信息
University of Illinois at Urbana-Champaign, Urbana, IL, United States.
Michigan State University, East Lansing, MI, United States.
出版信息
Methods Enzymol. 2017;582:137-169. doi: 10.1016/bs.mie.2016.10.036. Epub 2016 Dec 14.
Abstract
We describe the design, construction, and application of an instrument combining dual-trap, high-resolution optical tweezers and a confocal microscope. This hybrid instrument allows nanomechanical manipulation and measurement simultaneously with single-molecule fluorescence detection. We present the general design principles that overcome the challenges of maximizing optical trap resolution while maintaining single-molecule fluorescence sensitivity, and provide details on the construction and alignment of the instrument. This powerful new tool is just beginning to be applied to biological problems. We present step-by-step instructions on an application of this technique that highlights the instrument's capabilities, detecting conformational dynamics in a nucleic acid-processing enzyme.
摘要
我们描述了一种结合双阱、高分辨率光镊和共聚焦显微镜的仪器的设计、构建及应用。这种混合仪器能够在进行单分子荧光检测的同时实现纳米级机械操作与测量。我们阐述了在保持单分子荧光灵敏度的同时最大化光阱分辨率所面临的挑战及相应的通用设计原则，并提供了该仪器的构建与校准细节。这种强大的新工具才刚刚开始应用于生物学问题研究。我们逐步介绍了这项技术的一个应用实例，突出了该仪器的功能，即检测一种核酸加工酶中的构象动力学。
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