Correlative Light and Electron Microscopy (CLEM) is a method of choice to demonstrate the localization of a protein in a given organelle while revealing the ultrastructure of the organelle. However, a long-lasting problem in CLEM is the mismatch of resolution between modalities. As it becomes more and more clear that most organelles show a sub-compartmentalization of their membranes into distinct micro-domains, the visualization of such micro-domains at the molecular level using CLEM is a potential strategy to elucidate the molecular basis underlying the ultrastructure of organelles.

We developed a workflow that combines multi-color Single Molecule Localization Microscopy (SMLM) and electron tomography on the very same sample. We applied this approach to study the compartmentalization of early endosomes in micro-domains. As proof of principle, we visualized Transferrin and EGF as models of cargo molecules that follow the recycling and degradative route, respectively. Using single molecule CLEM tomography, we could visualize Transferrin and EGF molecules in distinct morphological compartments within the same endosome (see figure). We could also demonstrate the localization of Rab5 in micro-domains on the globular membrane of the endosome and its exclusion from the tubular parts. Beyond endocytosis, this method can be applied to any biological question requiring both multi-molecule nanoscale localization and 3D ultra-structural information of the very same sub-cellular structure.

Figures:

« Back to The 16th European Microscopy Congress 2016

EMC Abstracts - https://emc-proceedings.com/abstract/multi-color-correlative-palmstorm-and-electron-tomography-reveals-micro-domain-organization-of-endosomes/