biostudies-literatureKong HYouth Innovation Promotion Association, CASNational Natural Science Foundation of ChinaNational Key Research and Development Program of China1218-1227https://www.ebi.ac.uk/biostudies/studies/S-EPMC8288996biostudies-literatureUnknown7(7)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 National science reviewGenetically encoded X-ray cellular imaging for nanoscale protein localization.PMC82889962139041420122052016YFA040090220162361167525121834007Xia KLi JTai RYan QZhang JHu JZhu YWang JKong HFan CShin HJWang LfalseGenetically encoded X-ray cellular imaging for nanoscale protein localization.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 2020-01-01T00:00:00Z2020 Jul2025-04-19T21:17:46.528Z2025-04-19T21:17:46.528ZS-EPMC82889963469214610.1093/nsr/nwaa055