In this work we present what we believe to be the first detection of serotonin along with it’s derivatives and substrate, in vivo using a noninvasive technique. Serotonin (5-HT) is uniquely amenable to being measured using CEST. 5HT, along with it’s derivative 5HIAA and its substrate tryptophan, have a amide proton which is shifted far outside (10.5ppm referenced to tetramethylsilane) the usual range of exchangeable protons (fig. 1), and 5-HT remains in the slow exchange regime over the range of physiological pH values ¹. 5-HT, 5-HIAA, and tryptophan concentrations in the brain range from 10-100µM ², and as a result cannot be directly and noninvasively measured. 5-HT is currently implicated in the patho-physiology of major depressive and anxiety disorders³, and it is the target of many pharmacological interventions such as mono amine oxidase inhibitors (MAOIs), and serotonin selective reuptake inhibitors (SSRIs)³. All experiments where conducted on a Bruker (Ettlingen, Germany) 70/30 Biospec MRI system. Phantom experiments: Chemical exchange rates of 5-HT phantoms of varying concentration (25mM, 15mM, 5mM, and 1mM) were measured using a continuous wave (CW) saturation prepared PRESS (repetition time/echo time=10,000/25ms, saturation time=4,800ms) sequence with variable saturation pulse powers (0.1-3.0µT) over a range of saturation offsets (-2,500Hz to 3,000Hz, with 20Hz spacing). T₁ and T₂ relaxation time constants of the phantoms were measured using an inversion prepared fast spin echo sequence (repetition time/echo time=10,000/42ms, FSE factor=8) and a CPMG sequence (repetition time/echo time spacing=10,000/5ms, number of echoes=128) respectively. The water line areas for each saturation time were fit using full Bloch equation, including chemical exchange, simulations. Measured T₁ and T₂ of the water pool were provided as known parameters. In total four parameters were fit: the parameters CEST exchange rate constant, Rex, proton fraction of the CEST pool, Mob, its longitudinal, T_1b and transverse, T_2b relaxation times. Phantom parameters were then used for in vivo data analysis. Animal experiments: CEST spectra were measured in 4 naïve rats and 5 rats treated with MAOIs and tryptophan (IP injection 20 mg/kg tranylcypromine followed by 100mg/kg L-tryptophan, animals were imaged once they started exhibiting behavioral symptoms of 5-HT overload ~1.5 hours)2, in order to increase brain 5-HT, before and after treatment. In vivo CEST experiments were carried out using a saturation prepared echo planar imaging sequence (repetition time/echo time=5,000/21ms, saturation time=4800ms, saturation offsets= -2,000Hz to 2,500Hz, with 20Hz spacing, saturation power=1.0µT). B₁ mapping before and after the CEST acquisition was performed using WASSR. Reference frames at a saturation offset frequency of 20 kHz were acquired every other. CEST spectra for each voxel were interpolated and shifted according to the B₁ maps and averaged for anatomical ROI (cortical GM, WM, hippocampus, and other subcortical GM). The average CEST spectra were fit using five Lorentzian peaks, to account for the direct effect, semi-solid MT, NOE, amide CEST, and amine CEST plus a sixth peak (5-HT) with fixed position and width determined by the phantom experiments. 5-HT CEST peak areas were compared with 5-HT concentrations were assessed ex-vivo using an enzyme-linked immunosorbent assay (ELISA) (Rocky Mountain Diagnostics, Colorado, USA).The exchange rate of the amide proton, R_ex on 5-HT (pH=7.4, T=37°) was measured. In phantoms the area of the CEST peak correlated with 5-HT concentration (R=0.94, p<0.01). Naïve animals showed no statistically significant change in the area of the 5-HT CEST peak between imaging sessions. Animals treated with MAOIs and tryptophan after the first imaging session showed a statistically significant increase (14±3% p<0.05) in the 5-HT CEST peak in the cortical gray matter ROI, consistent with the expected increase in brain 5-HT levels. The increase in cortical 5-HT CEST can be seen in individual animals as shown in figure 2. Data for both naïve and treated animals is summarized in figure 3. The area of the 5-HT CEST peak shows good linearity with ex vivo concentration of 5-HT. This work represents the first in vivo MR measurement of 5-HT along with its substrate, tryptophan, and derivative, 5-HIAA. We believe that this measurement will serve as an important biomarker in future studies investigating the roll of serotonin in major brain disorders.