A General Strategy to Develop Intramolecular Spirocyclic Boron Dipyrromethene Fluorophores for Self‐Blinking Super‐Resolution Imaging

Abstract Intramolecular spirocyclic fluorophores, with their controllable on‐off switching, have enabled diverse stimuli‐responsive probes and serve as cornerstones of self‐blinking super‐resolution imaging. However, their design has been largely restricted to Rhodamine frameworks, while boron dipyrromethene (BODIPY) dyes, renowned for their superior photostability, brightness, and near‐infrared tunability, remain underutilized due to the inherent rigidity of the indacene core. To overcome this limitation, we developed a general molecular engineering strategy to confer BODIPY with spirocyclic functionality: Introducing 2‐(hydroxymethyl)phenyl or 2‐carboxyphenyl groups at the meso ‐position as intramolecular nucleophiles, coupled with electron‐withdrawing groups at α / β ‐positions to modulate core electron density and enhance meso ‐carbon's electrophilicity. We present the first single‐crystal X‐ray structure of a spirocyclized BODIPY, unambiguously confirming the closed form and its blinking mechanism. By tuning this system, we established a clear pK a ‐structure‐activity relationship, enabling precise prediction and customization of the open‐closed equilibrium. The resulting spirocyclization‐capable BODIPYs exhibited exceptional brightness up to ∼87,122 M −1 cm −1 , and enable self‐blinking super‐resolution imaging of mitochondria and lysosomes in living cells under low‐power laser density (≥32 W cm −2 ) without requiring imaging‐enhancing buffers. This work pioneers the extension of intramolecular spirocyclization to the BODIPY scaffold, establishing a general design strategy for a new class of high‐performance, stimuli‐responsive probes.