Water is essential in maintaining cellular activity, from mediating reactions to promoting the shuttling of macromolecules. Spectroscopic studies have indicated that the H-bonding network of water is disrupted by the hydrophobicity of solutes, molecular crowding, and pressure, leading to distinct water structures with the hydration shells of biomacromolecules. Intracellular water is within a very crowded and confined environment and, thus, cannot be adequately described as bulk water; instead, it is expected to exhibit more order or structure due to the confined and crowded environment. Raman spectral imaging is a powerful tool to identify and characterize water throughout a cell, generating subcellular hydration maps. This label-free approach relies on intrinsic molecular vibrational signatures enabling us to obtain spatial information on water and other biomacromolecules in a single experiment. We provide direct evidence that intracellular water does not exhibit the same vibrational properties as bulk water, supporting the importance of considering the effects of confinement and crowding when studying biomolecular systems.
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