Microenvironmental Fingerprint of Maturing Tau Condensates from Spectral and Lifetime Phasor Analysis of a Solvatochromic Dye

Solvatochromic dyes respond to changes in their local environment through variations in both spectral properties and fluorescence lifetime. These responses arise from the excited-state behavior of the probe, but the sensitivity of each response to a given environment can differ. In this work, we developed a multimodal phasor-based framework for two-photon spectral and fluorescence lifetime imaging analysis. To validate the framework, we imaged the solvatochromic dye ACDAN in water, glycerol, and ethanol. ACDAN showed clear spectral differences among the three solvents, whereas its fluorescence lifetime separated water from both organic solvents but not glycerol from ethanol, indicating that the two observables respond differently to the same environmental change. We then applied ACDAN to a well-described model system, the maturation of liquid-liquid phase-separated condensates of the microtubule-binding, neurodegeneration-related protein Tau into later-stage assemblies. Across Tau samples incubated for 1, 4, and 48 h, a progressive decrease in the ACDAN spectral phasor angle θ and increase in its phase-derived lifetime τP suggested increasingly restricted dipolar relaxation in the probe environment. Differences among condensates were apparent by 4 h, whereas pronounced spatial heterogeneity within assemblies was observed at 48 h. Lastly, we evaluated the multimodal framework on Tau assemblies labeled with the fluorogenic probe Thioflavin T. As expected, this dye predominantly sensed its environment through its fluorescence lifetime, with comparatively limited spectral changes, whereas ACDAN reported microenvironmental changes in both coordinates. Together, this framework combines spectral and lifetime descriptors to characterize changes in the fluorescence response across Tau assembly maturation while retaining their spatial distribution.