Unveiling biomolecular condensate physiology and pathology using spectral and time-resolved fluorescence microscopy of DAN probes

Biomolecular condensates are central to cellular physiology and disease. Understanding their molecular organization and regulation requires methods that go beyond intensity-based fluorescence microscopy to capture the environmental variables that define phase behavior. In this review, we highlight the use of environment-sensitive di-methylaminonaphtalene (DAN) probes, combined with hyperspectral imaging and fluorescence lifetime imaging microscopy, analyzed using the phasor approach, as powerful strategies for probing macromolecular crowding, water dynamics, and condensate interfaces in vitro and in vivo. After outlining the principles of spectral and lifetime phasor analysis, we illustrate applications across distinct biological contexts. In vitro studies show how crowding and membrane wetting modulate condensate formation and lipid packing. In virology, dengue and Zika capsid proteins undergo liquid-liquid phase separation (LLPS) with RNA and membranes, with implications for nucleocapsid assembly. In neurobiology, phase transitions of aggregation-prone proteins can be monitored to uncover early steps in pathological condensation. Finally, in vivo studies of the zebrafish lens reveal how aquaporins regulate macromolecular crowding and lens transparency. Together, these examples demonstrate how DAN probes combined with phasor analysis extend the toolkit for studying condensates, connecting nanoscale solvent dynamics with mesoscopic phase behavior and physiological outcomes.