Understanding structural dynamics of biomolecules at the single-molecule level is vital to advancing our knowledge of molecular mechanisms. Currently, there are few techniques that can capture dynamics at the sub-nanometre scale and in physiologically relevant conditions. Atomic force microscopy (AFM)¹ has the advantage of analysing unlabelled single molecules in physiological buffer and at ambient temperature and pressure, but its resolution limits the assessment of conformational details of biomolecules². Here we present localization AFM (LAFM), a technique developed to overcome current resolution limitations. By applying localization image reconstruction algorithms³ to peak positions in high-speed AFM and conventional AFM data, we increase the resolution beyond the limits set by the tip radius, and resolve single amino acid residues on soft protein surfaces in native and dynamic conditions. LAFM enables the calculation of high-resolution maps from either images of many molecules or many images of a single molecule acquired over time, facilitating single-molecule structural analysis. LAFM is a post-acquisition image reconstruction method that can be applied to any biomolecular AFM dataset.

在单分子水平上了解生物分子的结构动力学对于提高我们对分子机制的了解至关重要。目前，很少有技术可以在亚纳米尺度和生理相关条件下捕捉动力学。原子力显微镜 (AFM) ^{1具有在生理缓冲液和环境温度和压力下分析未标记单分子的优势，但其分辨率限制了对生物分子2}构象细节的评估。在这里，我们介绍了定位 AFM (LAFM)，这是一种为克服当前分辨率限制而开发的技术。通过应用定位图像重建算法³为了在高速 AFM 和常规 AFM 数据中达到峰值位置，我们将分辨率提高到超出尖端半径设定的限制，并在天然和动态条件下解析软蛋白表面上的单个氨基酸残基。LAFM 能够从多个分子的图像或随时间获取的单个分子的多个图像计算高分辨率图，从而促进单分子结构分析。LAFM 是一种采集后图像重建方法，可应用于任何生物分子 AFM 数据集。