ABSTRACT

Molecular aggregation profoundly alters the optical properties of fluorophores, yet its behavior in dynamic biological environments remains insufficiently understood and rarely harnessed functionally. Here, we report a membrane‐controlled aggregation strategy based on plasma membrane‐targeted BODIPY dimers engineered to undergo tunable

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‐ and

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‐aggregation. By modulating the linker length, we establish a clear structure–aggregation relationship in which dimers form intramolecular

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‐aggregates in polar media, while two‐dimensional confinement within lipid bilayers promotes intermolecular aggregation with red‐shifted emission. Quantitative studies in model membranes reveal a threshold‐dependent transition toward aggregate formation above a probe/lipid ratio of 1/100, highlighting the lipid bilayer as an active supramolecular platform that enhances probe‐probe encounters and favors emissive excitonic states. In live cells, this membrane‐promoted

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‐aggregation generates spontaneous blinking behavior that enables dual‐channel single‐molecule localization microscopy without specialized imaging buffers. Mechanistic investigations using HaloTag constructs confirm that while intramolecular aggregation can occur, membrane‐driven intermolecular collisions strongly amplify

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‐aggregate formation. These findings demonstrate that biological membranes can serve as dynamic two‐dimensional reactors for excitonic coupling, and establish membrane‐induced

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‐aggregation of small‐molecule fluorophores as a functional and generalizable principle for bioimaging.