Super‐Resolution Imaging of Clickable Lipids With Lipid Expansion Microscopy (LExM)

Fluorescent imaging of cellular membranes is challenged by the size of lipid bilayers, which are smaller than the diffraction limit of light. Recently, expansion microscopy (ExM) has emerged as an approachable super‐resolution method that requires only widely accessible confocal microscopes. In this method, biomolecules of interest are anchored to hydrogel‐based, polymeric networks that are expanded through osmosis to physically separate and resolve features smaller than the diffraction limit of light. Whereas ExM has been employed for super‐resolution imaging of proteins, DNA, RNA, and glycans, the application of this method to the study of lipids is challenged by the requirement of permeabilization procedures that remove lipids and compromise the integrity of the membrane. Here, we describe our recently developed protocols for lipid expansion microscopy (LExM), a method that enables ExM of membranes without permeabilization. These detailed protocols and accompanying commentary sections aim to make LExM accessible to any experimentalist interested in imaging membranes with super‐resolution.