Cryo-Correlative Light and X-ray microscopies: Expanding the Intracellular Chemical Map

Abstract We introduce a powerful, integrated workflow that fuses cryo-optical fluorescence microscopy with cryogenic synchrotron radiation X-ray fluorescence nanoimaging to unlock unprecedented nanoscale insights into cellular ultrastructure and composition. Our method delivers sharp 2D and 3D visualizations enabling simultaneous elemental mapping, nanoparticle tracking, and imaging of mitochondrial features via a luminescent cyclometalated iridium complex. We further demonstrated that combining well-chosen molecular probes possessing different heavy elements (e.g. rhenium, iridium, bromine and iodine) allows elemental multiplex “painting” of different organelle to provide X-ray fluorescent elemental contrast of some intracellular structure. By eliminating the need for separate sample preparations, this streamlined approach maximizes limited synchrotron beamtime and dramatically accelerates data acquisition, setting a new benchmark for advanced cryo-nanoscale imaging studies. Competing Interest Statement The authors have declared no competing interest.