Correlative X-ray imaging and fluorescence microscopy in hydrated environments

Imaging cellular organization in its native state is essential for understanding how the molecular architecture governs biological function. Fluorescence microscopy is one of the pivotal techniques that provides molecular specificity for imaging in real space, but is limited to labeled components. In contrast, X-rays are sensitive to electron density variations, enabling label-free structural characterization in reciprocal space. In particular, scanning small-angle X-ray scattering (SAXS) combines information from real and reciprocal space and enables access to intact cells owing to the high penetration power of X-rays. To harness the complementary strengths of these approaches, we developed a correlative imaging platform integrating a modular, compact, and beamline-compatible fluorescence microscope with scanning SAXS. The system enables fast sequential imaging of the same cellular regions under hydrated, near-native conditions using a dedicated microfluidic chamber. Applying this approach to keratin bundles in epithelial cells and force-generating actomyosin in cardiomyocytes, we demonstrate good agreement between fluorescence and SAXS-derived orientation maps. This result shows that label-free SAXS captures cytoskeletal organization throughout the cell, and can be directly linked to specific molecular information under physiologically relevant conditions. Our work establishes a general strategy for multimodal imaging of cellular architecture and enables studies of living cells under drugs and chemical manipulation.