PROJECT SUMMARY In the past, nearly all clinical and research proton magnetic resonance spectroscopy (MRS) studies of human brain metabolism have focused on the resonances upfield (UF) from the water signal. However, over the last few years it has been demonstrated that there are signals downfield (DF) from water that can be visualized using appropriate techniques, which may contain important metabolic information. We have recently shown that it is possible to map these signals throughout the human brain at 3T using 3D magnetic resonance spectroscopic imaging (MRSI). Furthermore, the clinical applicability of this technique would be significantly enhanced if it were possible to simultaneously map both UF and DF peaks at the same time. In this proposal, a new pulse sequence will be developed for DF-MRSI which will have improved sensitivity and excitation profiles compared to our current approach. Rigor and reproducibility will be carefully assessed; the sensitivity and reproducibility of this new approach will be compared to our previously developed DF-MRSI methodology in 10 healthy subjects scanned at two time points. In addition, assignment of DF metabolite peaks will be investigated in detail using multiple metabolite solutions scanned under carefully controlled physiological conditions. This novel pulse sequence may also be used for UF-MRSI acquisitions. Reproducibility and congruence with conventional UF-MRSI acquisition methods will be established, again by the study of 10 healthy volunteers at two time points. Finally, simultaneous UF- and DF-MRSI will be implemented with optimal sensitivity for both sides of the spectrum. Again, sensitivity and reproducibility will be compared to sequential UF- and DF-MRSI measurements made in the same total scan time in 10 healthy subjects at two time points. In parallel with the pulse sequence development and data acquisition aims, we will also continue to develop and disseminate our MRSI post-processing methods as part of our open source ‘Osprey’ software package, which is available to the clinical neuroscience and neuroimaging communities.