Target-switchable molecular imaging platform for 3D protein mapping in single cells by synchrotron radiation hard X-ray nanotomography

Developing an accessible and scalable strategy for three-dimensional (3D) protein imaging with hard X-ray nanotomography at the single-cell level remains a major analytical challenge due to the lack of intrinsic molecular specificity. Here, we report a target-switchable nanoprobe system based on biotinylated metal nanoparticles (BioMNs) that enables dual-modal fluorescence and hard X-ray imaging of specific proteins in intact single cells. In this modular architecture, the metal nanoprobes serve as universal X-ray signal modules, while molecular specificity is introduced through streptavidin-mediated coupling to biotinylated antibodies, thereby decoupling molecular recognition from signal generation. Combined with synchrotron radiation hard X-ray nanotomography (SR-HXT), this platform allows 3D visualization of diverse protein targets by simply replacing the primary antibody, eliminating the need for customized probe synthesis for each new target. As a validation of its versatility, membrane-associated HER2 and nuclear Ki67 were imaged as representative targets, revealing distinct spatial distributions and nanoscale heterogeneity in intact cells. This target-switchable approach overcomes the constraints of conventional single-target probes and establishes a correlative fluorescence–X-ray workflow for scalable 3D molecular imaging at the single-cell level.