Summary Water is one of the three major constituents of dental hard tissues, but the structure of water and how water interacts with other components in dental hard tissues during teeth formation, degradation, and lesion remain unclear. Nuclear magnetic spectrometry (NMR) and infrared absorption (IR) are the methods that can directly probe water from biological tissues. However, their performances are limited because NMR only measures the average effect of the sample, while IR requires special preparations to tissues like teeth and could alter the properties of the tissues. Recently, we demonstrated that fluorescence compressed Raman spectroscopy has the capability of probing water in dental hard tissues and showed that the spectral profiles of water are dramatically different between dentin and enamel. The profiles varied even within enamel at different testing spots. Those results indicate that Raman spectroscopy may provide additional information about the role of water in dental hard tissues with details that may impact the understanding of the mechanisms of dental problems. The major aim of the proposed project is to use Raman spectroscopy to identify how the changes in water content and structure in dental hard tissues may correlate with dental tissue pathologies. Aim 1 will determine the best available Raman spectroscopy configurations for probing water contents in dental hard tissues. Three common fluorescence suppression approaches, i.e., near-infrared excitation combining InGaAs detector, UV resonance Raman, and time-gated Raman spectroscopy will be compared to determine how the signal to noise ratio of the water-related Raman signals can be maximumly enhanced. We expect to increase the system sensitivity by one order or more. Aim 2 will investigate the changes in water amount and structure changes reflected by Raman spectroscopy and identify if these changes are correlated with other chemical/physical changes in dental hard tissues. Teeth with various degrees of demineralization and decomposing of organics will be prepared to show deferent levels of chemical compositional changes. Raman spectra will be acquired from these simulated samples and used as references for probing compositional variations in teeth with naturally developed caries. Raman spectroscopy will also be combined with micro-mechanical testing to identify correlations between water contents and structure change and mechanical property changes.