BPS2026 - Revealing biophysical mysteries of protein condensation in biomimetic systems using nanoscale spectroscopy and microscopy

Liquid-liquid phase separation (LLPS) is recognized as a fundamental principle underlying the formation of membrane-less organelles, orchestrating biochemical reactions in living cells. Yet, the molecular rules that govern condensate organization in vivo remain poorly understood due to the complexity and limited experimental control in cellular environments. Here, we apply advanced fluorescence methods to the characterization of tuneable biomimetic protein condensates to dissect how spatial confinement, macromolecular crowding and local physicochemical properties modulate protein condensation, compartmentalization and reaction dynamics. Segmented fluorescence correlation spectroscopy (FCS) revealed striking probe-dependent dynamics in the inside and outside of the protein condensates across very different temporal regimes: GFP behaved as an inert tracer, whereas Alexa showed interaction with BSA and lysozyme, yielding markedly distinct diffusion regimes. This demonstrates how segmented FCS can disentangle multiple dynamic scales while also highlighting how probe choice reshapes condensate behavior. Spectral phasor analysis of the environment-sensitive dye ACDAN indicated possible interfacial signatures, which might reflect polarity and hydration gradients at condensate boundaries, and could hint at nanoscale heterogeneity. To this end, sequential lambda scans and simultaneous 4-channel modalities (raster and line scan) were employed to assess the effect of temporal resolution on spectral imaging. To extend this framework, we are integrating fluorescence lifetime imaging microscopy (FLIM) and super-resolution techniques, including image scanning microscopy (ISM), stimulated emission depletion (STED), and SPLIT-PIN (separation of photons by lifetime tuning with adjustable pinhole size), to directly resolve nanoscale structural features. Our findings establish a powerful spectroscopic and microscopic toolkit for probing LLPS dynamics, advancing the integration of structure, function, and dynamics in protein condensates, and setting the stage for investigations into their roles in health and disease. (Funded by PRIN 2022 PNRR - Project: LLIPS P20228CCLL.)