Genetically Encoded APEX2 Enables Metal-Enhanced X-ray Contrast for Energy-Selective Imaging in Complex Tissues

X-ray microscopy offers deep tissue penetration and high spatial resolution, but its application to genetically targeted imaging remains limited by the lack of probes capable of generating sufficient and selective X-ray contrast in complex biological tissues. Here we report a genetically encoded X-ray contrast generation strategy that combines AAV-mediated expression of engineered peroxidase APEX2, enzymatic signal amplification through diaminobenzidine (DAB) deposition, and energy-selective synchrotron X-ray imaging. Optimization of Cre-dependent AAV expression and tissue fixation enabled efficient conversion of gene expression into localized X-ray-absorbing deposits. We further identified tissue-background absorption as a major factor limiting the analytical performance of conventional DAB-based X-ray imaging in dense brain regions. To address this challenge, nickel-enhanced DAB (EDAB) was introduced to generate metal-containing reaction products with characteristic absorption properties. Imaging near the Ni L-edge substantially improved signal discrimination between reporter-labeled neurons and surrounding tissue, overcoming the contrast limitations observed with conventional DAB imaging. This work establishes a genetically encoded and energy-selective X-ray contrast-generation platform that integrates reporter engineering, signal amplification, and absorption-edge-guided detection, providing a general analytical framework for targeted X-ray imaging in complex biological systems.