Biomolecular Condensates, Crowding, and Membranes: The Role of Water Revealed with Advanced Fluorescence Microscopy and Phasor Analysis.

The intracellular milieu is densely packed with macromolecules, creating a confined space for water molecules. The influence of macromolecular crowding on water activity impacts cell physiology and homeostasis, as well as the behavior of biomolecules and their supramolecular structures. In this context, understanding how water dynamics can be regulated could have profound implications for the tuning of the emergent properties of membrane-bound and membraneless organelles at the mesoscale.In this chapter, we discuss how the application of hyperspectral imaging (HSI) and fluorescence lifetime microscopy (FLIM) of the nano-environment sensors ACDAN and LAURDAN can be exploited to study hydration and water dynamics in cells and biomimetic systems. We will highlight the role of water in development, at the interface of membranous organelles, and in protein phase separation processes. Water plays a critical role in modulating physiology in all cases, providing a link for coupling protein and membrane structures. Combining fluorescence spectroscopy with advanced imaging constitutes a powerful approach for measuring and understanding water dynamics in cell physiology and disease.