Confinement-Free Wide-Field Ratiometric Tracking of Single Fluorescent Molecules
Confinement-Free Wide-Field Ratiometric Tracking of Single Fluorescent Molecules
复制标题
DOI：
10.1016/j.bpj.2019.10.033
复制
发表时间：
2019-12-03
影响因子：
3.4
通讯作者：
Kapanidis，Achillefs N.
中科院分区：
文献类型：
--
作者：
Gilboa，Barak;Jing，Bo;Kapanidis，Achillefs N.
在过去的二十年里，单分子荧光已经在阐明生物分子的相互作用和动力学方面发挥了重要作用。单分子荧光实验通常依赖于两种检测几何结构之一，共聚焦点检测或宽视场区域检测，通常采用全内反射荧光（TIRF）格式。然而，这些技术中的每一种都存在限制其应用的基本缺点。在这项工作中，我们提出了一种新的技术，解决方案宽场成像（SWiFi）的扩散分子，作为替代现有的方法。SWiFi是对现有物镜型TIRF显微镜的简单扩展，它允许宽视场观察快速扩散的分子，直到单个荧光团，而不需要将分子束缚在表面。我们证明，SWiFi使高通量比率测量与数千个单独的数据点，每分钟的双链DNA标准（dsDNA）样品含有福斯特共振能量转移对。我们进一步显示的能力，SWiFi的流动性和比率表征的荧光纳米金刚石，DNA霍利迪路口，和蛋白质-DNA相互作用的报告。SWiFi的高通量，快速扩散物种的比率测量的能力，使其成为一个有价值的工具，单分子研究社区之间的桥梁共聚焦和TIRF检测几何形状在一个简单而有效的方式。
Single-molecule fluorescence has been highly instrumental in elucidating interactions and dynamics of biological molecules in the past two decades. Single-molecule fluorescence experiments usually rely on one of two detection geometries, either confocal point-detection or wide-field area detection, typically in a total internal reflection fluorescence (TIRF) format. However, each of these techniques suffers from fundamental drawbacks that limit their application. In this work, we present a new technique, solution wide-field imaging (SWiFi) of diffusing molecules, as an alternative to the existing methods. SWiFi is a simple extension to existing objective-type TIRF microscopes that allows wide-field observations of fast-diffusing molecules down to single fluorophores without the need of tethering the molecules to the surface. We demonstrate that SWiFi enables high-throughput ratiometric measurements with several thousands of individual data points per minute on double-stranded DNA standard (dsDNA) samples containing Förster resonance energy transfer pairs. We further display the capabilities of SWiFi by reporting on mobility and ratiometric characterization of fluorescent nanodiamonds, DNA Holliday junctions, and protein-DNA interactions. The ability of SWiFi for high-throughput, ratiometric measurements of fast-diffusing species renders it a valuable tool for the single-molecule research community by bridging between confocal and TIRF detection geometries in a simple and efficient way.