Chang Jerry C, Rosenthal Sandra J
Department of Chemistry, Vanderbilt University, Nashville, TN, USA.
Methods Mol Biol. 2013;1026:71-84. doi: 10.1007/978-1-62703-468-5_6.
Real-time microscopic visualization of single molecules in living cells provides a molecular perspective of cellular dynamics, which is difficult to be observed by conventional ensemble techniques. Among various classes of fluorescent tags used in single-molecule tracking, quantum dots are particularly useful due to their unique photophysical properties. This chapter provides an overview of single quantum dot tracking for protein dynamic studies. First, we review the fundamental diffraction limit of conventional optical systems and recent developments in single-molecule detection beyond the diffraction barrier. Second, we describe methods to prepare water-soluble quantum dots for biological labeling and single-molecule microscopy experimental design. Third, we provide detailed methods to perform quantum dot-based single-molecule microscopy. This technical section covers three protocols including (1) imaging system calibration using spin-coated single quantum dots, (2) single quantum dot labeling in living cells, and (3) tracking algorithms for single-molecule analysis.
对活细胞中的单分子进行实时显微可视化，可提供细胞动力学的分子视角，而这是传统整体技术难以观察到的。在用于单分子追踪的各类荧光标签中，量子点因其独特的光物理性质而特别有用。本章概述了用于蛋白质动态研究的单量子点追踪。首先，我们回顾传统光学系统的基本衍射极限以及超越衍射屏障的单分子检测的最新进展。其次，我们描述用于生物标记的水溶性量子点的制备方法和单分子显微镜实验设计。第三，我们提供执行基于量子点的单分子显微镜的详细方法。本技术部分涵盖三个方案，包括（1）使用旋涂单量子点进行成像系统校准，（2）活细胞中的单量子点标记，以及（3）用于单分子分析的追踪算法。
