Bailey Danielle M, Kovtun Oleg, Rosenthal Sandra J
Department of Chemistry, Vanderbilt University, Nashville, TN, 37235, USA.
Interdisciplinary Materials Science Program, Vanderbilt University, Nashville, TN, 37235, USA.
Methods Mol Biol. 2017;1570:165-177. doi: 10.1007/978-1-4939-6840-4_11.
Single particle tracking (SPT) experiments have provided the scientific community with invaluable single-molecule information about the dynamic regulation of individual receptors, transporters, kinases, lipids, and molecular motors. SPT is an alternative to ensemble averaging approaches, where heterogeneous modes of motion might be lost. Quantum dots (QDs) are excellent probes for SPT experiments due to their photostability, high brightness, and size-dependent, narrow emission spectra. In a typical QD-based SPT experiment, QDs are bound to the target of interest and imaged for seconds to minutes via fluorescence video microscopy. Single QD spots in individual frames are then linked to form trajectories that are analyzed to determine their mean square displacement, diffusion coefficient, confinement index, and instantaneous velocity. This chapter describes a generalizable protocol for the single particle tracking of membrane neurotransmitter transporters on cell membranes with either unmodified extracellular antibody probes and secondary antibody-conjugated quantum dots or biotinylated extracellular antibody probes and streptavidin-conjugated quantum dots in primary neuronal cultures. The neuronal cell culture, the biotinylation protocol and the quantum dot labeling procedures, as well as basic data analysis are discussed.
单粒子追踪（SPT）实验为科学界提供了关于单个受体、转运体、激酶、脂质和分子马达动态调控的宝贵单分子信息。SPT是总体平均方法的一种替代方法，在总体平均方法中，异质运动模式可能会丢失。量子点（QD）由于其光稳定性、高亮度以及尺寸依赖性窄发射光谱，是SPT实验的优秀探针。在典型的基于量子点的SPT实验中，量子点与感兴趣的靶标结合，并通过荧光视频显微镜成像数秒至数分钟。然后将各个帧中的单个量子点光斑连接起来形成轨迹，对这些轨迹进行分析以确定其平均平方位移、扩散系数、限制指数和瞬时速度。本章描述了一种可推广的方案，用于在原代神经元培养物中，使用未修饰的细胞外抗体探针和二抗偶联的量子点或生物素化的细胞外抗体探针和链霉亲和素偶联的量子点，对细胞膜上的膜神经递质转运体进行单粒子追踪。文中讨论了神经元细胞培养、生物素化方案和量子点标记程序，以及基本数据分析。
