Efficient murine cardiac phenotyping by combining synchrotron-based phase-contrast micro-CT, histology, immunofluorescence and spatial transcriptomics

1.Congenital heart disease is commonly studied using genetically modified mouse models, but characterization of complex three-dimensional (3D) cardiac anomalies remains technically challenging. Histology of fixed paraffin-embedded samples, typically used for structural and molecular analysis, is limited to two dimensions (2D). Synchrotron radiation-based phase-contrast micro-computed tomography (SRPC-{micro}CT) enables rapid, high-resolution, 3D imaging but had yet to be fully integrated with molecular tissue analysis. Importantly, SRPC-{micro}CT is nondestructive and compatible with paraffin-embedded tissue, potentially allowing integration with downstream molecular analyses such as multiplexed spatial transcriptomic profiling on tissue sections. Here, we present a pipeline combining SRPC-{micro}CT with complementary molecular approaches for rapid and accurate phenotyping of mouse hearts and for investigating disease mechanisms. We demonstrate the successful integration of high-resolution 3D imaging with spatial transcriptomics, fluorescent stainings, and histochemistry. Notably, all 2D modalities were applied sequentially to a single tissue section and registered within the 3D volume. This multimodal framework provides a powerful approach for linking structural and molecular information that is broadly applicable across biomedical research.