Fluorescence microscopy, with its molecular specificity, is one of the major characterization methods used in the life sciences to understand complex biological systems. Super-resolution approaches^1,2,3,4,5,6 can achieve resolution in cells in the range of 15 to 20 nm, but interactions between individual biomolecules occur at length scales below 10 nm and characterization of intramolecular structure requires Ångström resolution. State-of-the-art super-resolution implementations^{7,8,9,10,11,12,13,14} have demonstrated spatial resolutions down to 5 nm and localization precisions of 1 nm under certain in vitro conditions. However, such resolutions do not directly translate to experiments in cells, and Ångström resolution has not been demonstrated to date. Here we introdue a DNA-barcoding method, resolution enhancement by sequential imaging (RESI), that improves the resolution of fluorescence microscopy down to the Ångström scale using off-the-shelf fluorescence microscopy hardware and reagents. By sequentially imaging sparse target subsets at moderate spatial resolutions of >15 nm, we demonstrate that single-protein resolution can be achieved for biomolecules in whole intact cells. Furthermore, we experimentally resolve the DNA backbone distance of single bases in DNA origami with Ångström resolution. We use our method in a proof-of-principle demonstration to map the molecular arrangement of the immunotherapy target CD20 in situ in untreated and drug-treated cells, which opens possibilities for assessing the molecular mechanisms of targeted immunotherapy. These observations demonstrate that, by enabling intramolecular imaging under ambient conditions in whole intact cells, RESI closes the gap between super-resolution microscopy and structural biology studies and thus delivers information key to understanding complex biological systems.

荧光显微镜以其分子特异性，是生命科学中用于了解复杂生物系统的主要表征方法之一。超分辨率方法^{1、2、3、4、5、6}可以在细胞中实现 15 至 20 nm 范围内的分辨率，但单个生物分子之间的相互作用发生在 10 nm 以下的长度尺度上，分子内结构的表征需要埃级分辨率。最先进的超分辨率实现^{7,8,9,10,11,12,13,14}在某些体外条件下已证明空间分辨率低至 5 nm，定位精度为 1 nm。然而，这种分辨率并不能直接转化为细胞实验，而且埃级分辨率迄今为止尚未得到证实。在这里，我们介绍了一种 DNA 条形码方法，即通过顺序成像 (RESI) 提高分辨率，该方法使用现成的荧光显微镜硬件和试剂将荧光显微镜的分辨率提高到埃级。通过以 >15 nm 的中等空间分辨率连续成像稀疏目标子集，我们证明整个完整细胞中的生物分子可以实现单蛋白分辨率。此外，我们通过实验以埃级分辨率解析了 DNA 折纸中单个碱基的 DNA 主链距离。我们在原理验证演示中使用我们的方法来绘制未经处理和药物处理的细胞中免疫治疗靶标 CD20 的原位分子排列图，这为评估靶向免疫治疗的分子机制提供了可能性。 这些观察结果表明，通过在环境条件下对整个完整细胞进行分子内成像，RESI 缩小了超分辨率显微镜和结构生物学研究之间的差距，从而提供了理解复杂生物系统的关键信息。