Scientists or clinicians interested in oxidative stress, reactive oxygen species, and CEST MRI. Oxidative stress due to elevated reactive oxygen species (ROS) is a critical contributor in various processes including normal aging, Alzheimer's disease, stroke, cardiac arrest, cancer, and diabetes (1-3). Direct and non-invasive approaches for quantifying tissue ROS will provide useful information in early diagnosis, disease stratification, and treatment response. Given that ROS has an extremely short lifetime (~10^-9 s for hydroxyl radicals) (4), and its in-vivo concentration is in µM level(5), it has been difficult to perform direct measurement. Currently there is no direct and non-invasive method to assess ROS production (6-9). In this study, we aim at developing a fully non-invasive method to measure tissue ROS concentration based on endogenous MRI contrasts including the chemical exchange saturation transfer (CEST) contrast that origins from exchangeable protons associated with specific metabolites. We investigated CEST MRI properties of ROS produced by adding hydrogen peroxide (H₂O₂) into ex-vivo tissues. Fresh egg white tissues (n=5) treated with various concentrations of H₂O₂ (0.25, 0.1, 0.05, and 0.025 v/v%) were scanned at different time points(0.5, 1, and 3 hours) at room temperature. Fluorescence studies using the hydroxyphenyl fluorescein (HPF) dye were performed to measure ROS production from the samples treated within 1 hour. CEST Z-spectra were collected on a horizontal 9.4-T MRI scanner using a customized sequence with a frequency-selective rectangle saturation pulse (B₁=50 Hz, 3 s) followed by a Fast Low-Angle Shot (FLASH) readout. The entire Z-spectra contained 49 saturation offsets ranging from -5 to 5 ppm with steps of 0.25 ppm, and at ±10, ±20, ±50, and ±100 ppm. CEST asymmetry effect was computed at 3.5 ppm. In a separate study, the proton exchange rates of untreated and treated (0.1 v/v% H₂O₂) egg white samples (n=4) were assessed based on the method previously described (10). The steady-state CEST images were acquired with a 4 s long saturation pulse at various saturation amplitudes B₁ from 50 up to 450 Hz. Data were fitted with a polynomial function to determine the amplitude leading to the maximal steady-state CEST contrast. In addition, T₁, T₂, and pH were measured.Compared to untreated egg white samples, H₂O₂ treatment modulated the entire CEST Z-spectra (Figure 1A), resulting in reductions in the CEST contrast. The CEST contrast reduction was proportional to H₂O₂ concentration (Figure 1B). Fluorescence studies showed that ROS production was also proportional to H₂O₂ treatment concentrations (Figure 1C). Interestingly, the required saturation amplitude for the maximum steady-state CEST contrast was much higher for the treated samples than the untreated, indicating that ROS greatly enhances proton exchange rate (Figure 2). Since CEST contrast relies on slow-to-intermediate proton exchange, we believe that ROS reduces CEST contrast by substantially raising the proton exchange rate. In addition, pH measurements showed only minor changes between baseline and at 1 hour post-treatment with 0.25 v/v% of H2O2 (pH = 9.00 ± 0.01 vs. 8.92 ± 0.01). These suggest that ROS likely serves as an independent exchange modulator from pH. We also found that T₁ relaxation time in egg white reduced due to H₂O₂ treatments, while the reduction in T₂ relaxation time was negligible (Figure 3). This indicates that ROS has a strong paramagnetic effect that can be detected by T₁-weighted MRI. In contrast, intact H₂O₂ (in PBS) does not affect T₁ significantly. At 3 hours post treatment, CEST contrast recovered almost back to the pre-treatment level (Figure 4), indicating all the H₂O₂ was converted to ROS within this time. We modeled the dynamics of CEST contrast as an exponential decay function and estimated that the ROS concentration (with 0.025 v/v% H2O2 at 1 hour post-treatment) was about 50 µM, which is within the in-vivo pathological or physiological range (5, 11, 12). We are currently working toward the in-vivo applications of this technique.In summary, we have observed that ROS is paramagnetic. ROS also greatly enhances proton exchange, independent to pH, leading to reduced CEST contrast in general. We have showed that CEST combined with T₁-weighted contrast has the potential to improve the specificity for imaging ROS or oxidative stress in tissue. This novel MRI technique may serve as a longitudinal imaging tool for biochemical analysis of ROS production in ex-vivo tissue or other experimental set-ups. It may also allow for non-invasively quantifying and mapping ROS production in vivo with high temporal and spatial resolutions.