Super-resolution microscopy techniques have pushed the limit of optical imaging to unprecedented spatial resolutions. However, one of the frontiers in nanoscopy is its application to intact living organisms. Here we describe the implementation and application of super-resolution single-particle tracking photoactivated localization microscopy (sptPALM) to probe single-molecule dynamics of membrane proteins in live roots of the model plant Arabidopsis thaliana. We first discuss the advantages and limitations of sptPALM for studying the diffusion properties of membrane proteins and compare this to fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS). We describe the technical details for handling and imaging the samples for sptPALM, with a particular emphasis on the specificity of imaging plant cells, such as their thick cell walls or high degree of autofluorescence. We then provide a practical guide from data collection to image analyses. In particular, we introduce our sptPALM_viewer software and describe how to install and use it for analyzing sptPALM experiments. Finally, we report an R statistical analysis pipeline to analyze and compare sptPALM experiments. Altogether, this protocol should enable plant researchers to perform sptPALM using a benchmarked reproducible protocol. Routinely, the procedure takes 3–4 h of imaging followed by 3–4 d of image processing and data analysis.

超分辨率显微镜技术将光学成像的极限推向了前所未有的空间分辨率。然而，纳米显微镜的前沿之一是它在完整生物体中的应用。在这里，我们描述了超分辨率单粒子跟踪光激活定位显微镜 (sptPALM) 的实现和应用，以探测模型植物拟南芥活根中膜蛋白的单分子动力学. 我们首先讨论 sptPALM 在研究膜蛋白扩散特性方面的优势和局限性，并将其与光漂白后的荧光恢复 (FRAP) 和荧光相关光谱 (FCS) 进行比较。我们描述了处理和成像 sptPALM 样品的技术细节，特别强调成像植物细胞的特异性，例如它们的厚细胞壁或高度的自发荧光。然后，我们提供从数据收集到图像分析的实用指南。特别是，我们介绍了我们的 sptPALM_viewer 软件，并描述了如何安装和使用它来分析 sptPALM 实验。最后，我们报告了一个 R 统计分析管道来分析和比较 sptPALM 实验。共，该协议应使植物研究人员能够使用基准可重现协议执行 sptPALM。通常，该过程需要 3-4 小时的成像，然后是 3-4 天的图像处理和数据分析。