Chemical exchange saturation transfer (CEST) MRI is a relatively new technology, and has become a vigorous research field where new contrast mechanisms are constantly being developed. Examples of this technology include the measurement of in vivo metabolite, neurotransmitter, and contrast agent concentrations, the measurement of in vivo pH, and the development of biomarkers for disorders such as glioma, osteoarthritis, muscle dysfunction, and type 2 diabetes. CEST contrast promises the ability to measure small concentrations of molecules via the large bulk water pool, hence with largely enhanced sensitivity. The major obstacles to widespread implementation of CEST MRI remain (1) the appearance of magnetization transfer (MT) asymmetry, and (2) the susceptibility to B0 field inhomogeneities, both of which can lead to false positives and false negatives. MT asymmetry arises from exchangeable protons or through-space magnetization transfer processes between immobile macromolecular structures and bulk water, and is a strong effect in many tissues including cartilage, muscle, and the brain. This proposal aims at 'decoupling' MT asymmetry from CEST entirely and directly, via the implementation of a new strategy: the investigators have recently discovered that broad resonances in tissues can be efficiently saturated via properly-placed simultaneous two-frequency irradiation and that MT asymmetry can be saturated efficiently and uniformly in CEST (2frequCEST). The MT saturation process does not rely on subtraction or the linearity of the MT effect. In addition, problem (2) will be addressed by using adapted B0 correction protocols with this new methodology. The primary testing ground of this approach will be the glycosaminoglycan (GAG) assessment in cartilage. This strategy is in itself very important for the development of early markers for osteoarthritis. The investigators have acquired considerable expertise in this field, both with gagCEST and sodium MRI methodology. The overall hypothesis is that two-frequency CEST provides better diagnostic ability than conventional CEST due to efficient elimination of MT asymmetry. The correlation will be assessed via MR spectroscopy (N-acetyl signal of GAG) and sodium MRI. The strategy is broken down into three specific aims: (1) Develop, quantify, and simulate 2frequCEST ex vivo, (2) Validate 2frequCEST with sodium content via microimaging ex vivo (sodium MR has been shown to be an efficient marker for GAG concentration in vivo). (3) Validate 2frequCEST with fluid-suppressed 23Na MRI on 3T/7T scanners in vivo. Successful completion of this project will provide a powerful proton-based diagnostic tool for devising effective monitoring strategies for osteoarthritis, but also potentially for many other disorders, such as muscle dysfunction, type 2 diabetes, as well as for the in vivo monitoring of metabolism and other diagnostic tasks where isolation of CEST from MT asymmetry is required.