Characterization of Intra/Extracellular Water States Probed by Ultrabroadband Multiplex Coherent Anti-Stokes Raman Scattering (CARS) Spectroscopic Imaging.

Detailed knowledge of the water status in living organisms is crucial for understanding their physiology and pathophysiology. Here, we developed a technique to spectroscopically image water at high resolution using ultrabroadband multiplex coherent anti-Stokes Raman scattering (CARS) microscopy equipped with a supercontinuum light source. This system allows for the visualization of a wide spectrum of CARS signals from the fingerprint to the end of O-H stretching at a spectral resolution of ∼10 cm-1. Application of the system to living mammalian cells revealed a spectral red shift of the O-H stretching vibrational band inside compared to outside the cells, suggesting the existence of stronger hydrogen bonds inside the cells. Furthermore, potential changes in spectra were examined by adding mannitol to the extracellular solution, which increases the osmolality outside the cells and thereby induces dehydration of the cells. Under this treatment, the red shift of the O-H stretching band was further enhanced, revealing the effects of mannitol on water states inside the cells. The methodology developed here should serve as a powerful tool for the chemical imaging of water in living cells in various biological and medical contexts.