Correlative X-ray imaging and fluorescence microscopy

Imaging the structural organization inside cells in their native state is essential for understanding how the arrangement and interactions among molecular components give rise to biological function. Fluorescence microscopy is one of the pivotal techniques that provides molecular specificity for imaging in real space, however, the technique is limited to labeled components. X-rays, on the contrary, are sensitive to electron density contrast and therefore to label-free samples, and probe structure 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 the X-rays. Combining both imaging modalities in a synergistic manner promises powerful tools for cellular imaging, but remains challenging, because of the differing requirements the complementary methods introduce. Here we present a correlative imaging platform that integrates a modular, compact and beamline-compatible fluorescence microscope with scanning SAXS, to enable fast sequential imaging of the identical cellular regions. We developed a dedicated microfluidics flow chamber enabling measurements under hydrated, near-native conditions. We demonstrate the utility of our methodology by investigating two different relevant cellular components, i.e., thick keratin bundles in epithelial cells that contribute to cell mechanics, and force-generating actomyosin in cardiomyocytes. Employing adapted data analysis methods, we find a good agreement between the fluorescence-derived and the SAXS-derived orientation maps. This result demonstrates that the label-free approach with SAXS captures cytoskeletal organization throughout the cell, and can be directly linked to specific molecular information provided by the complementary fluorescence imaging, in a physiologically relevant cellular environment. Our work establishes a general strategy for multimodal imaging of cellular architecture and opens ways to investigate living cells under the influence of drugs and chemical manipulation experiments.