Correlating STED and synchrotron XRF nano-imaging to explore metal functions in synapses

Zinc and copper are involved in neuronal differentiation and synaptic plasticity but the molecular mechanisms behind these processes are still elusive due in part to the difficulty of imaging trace metals together with proteins at the synaptic level. We correlate stimulated-emission-depletion microscopy (STED) of proteins and synchrotron X-ray fluorescence (XRF) imaging of trace metals, both performed with 40 nm spatial resolution, on primary rat hippocampal neurons. Synchrotron XRF was performed on the ID16A Nano-Imaging beamline at the European Synchrotron Radiation Facility. ID16A beamline provides a pink nanoprobe (ΔE/E≈1%) at two discrete energies: E = 17kev and 33.6 kev. For the correlative STED-SXRF experiments 17 keV was chosen, despite a larger focus at lower energy, as it is more efficient in exciting the XRF of the biologically relevant elements zinc and copper. The X-ray focus with dimensions of 35 nm (H) x 57 nm (V) provided a flux of 3.7 10¹¹ ph/s. Thanks to this experimental setup, we reveal the co-localization at the nanoscale of zinc and tubulin in dendrites with a molecular ratio of about one zinc atom per tubulin-αβ dimer. We observe the co-segregation of copper and F-actin within the nano-architecture of dendritic protrusions. These results indicate new functions for zinc and copper in the modulation of the synaptic cytoskeleton morphology, a mechanism associated to structural plasticity. From a methodological perspective, the combination of STED super-resolution microscopy and nano-SXRF imaging stands as a solid new tool for the identification of metalloproteins directly in cells by correlating cellular imaging methods at a supramolecular scale.