Optical mapping of biological water in single live cells by stimulated Raman excited fluorescence microscopy

Water is arguably the most common and yet least understood material on Earth. Indeed, the biophysical behavior of water in crowded intracellular milieu is a long-debated issue. Understanding of the spatial and compositional heterogeneity of water inside cells remains elusive, largely due to a lack of proper water-sensing tools with high sensitivity and spatial resolution. Recently, stimulated Raman excited fluorescence (SREF) microscopy was reported as the most sensitive vibrational imaging in the optical far field. Herein we develop SREF into a water-sensing tool by coupling it with vibrational solvatochromism. This technique allows us to directly visualize spatially-resolved distribution of water states inside single mammalian cells. Qualitatively, our result supports the concept of biological water and reveals intracellular water heterogeneity between nucleus and cytoplasm. Quantitatively, we unveil a compositional map of the water pool inside living cells. Hence we hope SREF will be a promising tool to study intracellular water and its relationship with cellular activities.