Spatial resolution defines the physical limit of microscopes for probing biomolecular localization and interactions in cells. Whereas synchrotron-based X-ray microscopy (XRM) represents a unique approach for imaging a whole cell with nanoscale resolution due to its intrinsic nanoscale resolution and great penetration ability, existing approaches to label biomolecules rely on the use of exogenous tags that are multi-step and error-prone. Here, we repurpose engineered peroxidases as genetically encoded X-ray-sensitive tags (GXET) for site-specific labeling of protein-of-interest in mammalian cells. We find that 3,3^′-diaminobenzidine (DAB) polymers that are in-situ catalytically formed by fusion-expressed peroxidases are visible under XRM. Using this new tag, we imaged the protein location associated with the alteration of a DNA-methylation pathway with an ultra-high resolution of 30 nanometers. Importantly, the excellent energy resolution of XRM enables multicolor imaging using different peroxidase tags. The development of GXET enlightens the way to nanoscopic imaging for biological studies.

中文翻译：

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用于纳米级蛋白质定位的基因编码 X 射线细胞成像

空间分辨率定义了用于探测细胞中生物分子定位和相互作用的显微镜​​的物理极限。基于同步加速器的 X 射线显微镜 (XRM) 由于其固有的纳米级分辨率和强大的穿透能力，代表了一种以纳米级分辨率对整个细胞进行成像的独特方法，而现有的标记生物分子的方法则依赖于使用外源性标签。步骤和容易出错。在这里，我们将工程过氧化物酶重新用作基因编码的 X 射线敏感标签 (GXET)，用于哺乳动物细胞中感兴趣蛋白质的位点特异性标记。我们发现 3,3 ^′由融合表达的过氧化物酶原位催化形成的二氨基联苯胺 (DAB) 聚合物在 XRM 下可见。使用这个新标签，我们以 30 纳米的超高分辨率对与 DNA 甲基化途径改变相关的蛋白质位置进行了成像。重要的是，XRM 出色的能量分辨率使使用不同过氧化物酶标签的多色成像成为可能。GXET 的发展为生物研究的纳米级成像开辟了道路。