Affiliation:
1. Department of Chemistry and Chemical Biology Cornell University Ithaca New York
2. Weill Institute for Cell and Molecular Biology Cornell University Ithaca New York
3. Current affiliation: Department of Chemistry University of New Hampshire Durham New Hampshire
Abstract
AbstractFluorescent 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. © 2024 Wiley Periodicals LLC.Basic Protocol 1: LExM of alkyne‐choline lipidsBasic Protocol 2: LExM of IMPACT‐labeled lipidsBasic Protocol 3: LExM of clickable cholesterolBasic Protocol 4: Determining the expansion factor
Funder
National Institutes of Health