Switchable Solvatochromic Probes for Live‐Cell Super‐resolution Imaging of Plasma Membrane Organization

Abstract Visualization of the nanoscale organization of cell membranes remains challenging because of the lack of appropriate fluorescent probes. Herein, we introduce a new design concept for super‐resolution microscopy probes that combines specific membrane targeting, on/off switching, and environment sensing functions. A functionalization strategy for solvatochromic dye Nile Red that improves its photostability is presented. The dye is grafted to a newly developed membrane‐targeting moiety composed of a sulfonate group and an alkyl chain of varied lengths. While the long‐chain probe with strong membrane binding, NR12A, is suitable for conventional microscopy, the short‐chain probe NR4A, owing to the reversible binding, enables first nanoscale cartography of the lipid order exclusively at the surface of live cells. The latter probe reveals the presence of nanoscopic protrusions and invaginations of lower lipid order in plasma membranes, suggesting a subtle connection between membrane morphology and lipid organization.