In Situ 3D-FLIM Imaging of Extracellular Microvesicles Dynamics in Alzheimer's Disease via a Coumarin-Based Probe.

Extracellular microvesicles (EMVs) derived from diseased parental cells carry disease-specific cargos that reflect pathological features, making them promising biomarkers for Alzheimer's disease (AD). However, real-time tracking of EMVs dynamics for early AD diagnosis is hampered by conventional fluorescent probes, which fail to selectively target EMVs and quantify intravascular-microenvironmental fluctuations inside vesicles. To address these challenges, we designed and synthesized a series of coumarin-based amphiphilic fluorescent probes with clog P values below 1 for EMVs labeling and systematically optimized their optical properties and viscosity sensitivity by adjusting the electron-acceptor and π-bridge structures. The optimized probe, CQS3, combines suitable electropositivity, hydrophilicity, and a twisted intramolecular charge transfer structure. This probe enables not only selectively staining of EMVs rather than apoptotic bodies or exosomes but also tracking of microenvironment viscosity changes of EMVs via fluorescence lifetime imaging. Using this probe, we have demonstrated for the first time that the viscosity of the EMVs microenvironment is not directly correlated with EMVs size and that EMVs undergo irregular viscosity fluctuations during normal dynamic processes such as fusion and fission. Most importantly, the viscosity of EMVs shows a significant and synchronous increase during the pathological progression of AD. This research not only paves the way for advanced probe design for detailed EMVs imaging but also lays the groundwork for exploring EMVs dynamics as a precise early diagnostic marker for AD.