Visualizing sub-organellar lipid distribution using correlative light and electron microscopy

Lipids and proteins compartmentalize biological membranes into nanoscale domains which are crucial for signaling, intracellular trafficking and many other cellular processes. Studying nanodomain function requires the ability to measure protein and lipid localization at the nanoscale. Current methods for visualizing lipid localization do not meet this requirement. Here, we introduce a correlative light and electron microscopy workflow to image lipids (Lipid-CLEM), combining near-native lipid probes and on-section labeling by click chemistry. This approach enables the quantification of relative lipid densities in membrane nanodomains. We find differential partitioning of sphingomyelin into intraluminal vesicles, recycling tubules, and the boundary membrane of the early endosome, representing a degree of nanoscale organization previously observed only for proteins. We anticipate that our Lipid-CLEM workflow will greatly facilitate the mechanistic analysis of lipid functions in cell biology, allowing for the simultaneous investigation of proteins and lipids during membrane nanodomain assembly and function.