Microscopía de expansión (ExM) de lípidos para imágenes de membranas de super-resolución

Lipid Expansion Microscopy (LExM) is a super-resolution imaging technique that enables nanoscale visualization of cellular membranes in cultured cells using confocal microscopy. The procedure relies on the preservation and anchoring of lipids into a swellable hydrogel that is physically expanded to increase resolution. By labeling lipids with alkyne functional groups and covalently anchoring these bioorthogonally labeled lipids into a hydrogel network via copper-catalyzed azide-alkyne cycloaddition (CuAAC) with a trifunctional probe, LExM facilitates direct, super-resolution imaging of membrane ultrastructure. This chapter presents a comprehensive protocol for (i) labeling of cellular membranes with phospholipid precursors and cholesterol analogs, (ii) click chemistry tagging with a trifunctional, fluorophore-bearing probe, (iii) hydrogel embedding, (iv) expansion, and (v) imaging. The method enables detailed membrane visualization at sub-diffraction resolution via 4-7.8x sample expansion, is compatible with standard fluorescence microscopy, and is adaptable to multiplexed imaging of protein-lipid colocalization.