D-glucose can be used as a natural biodegradable contrast agent for cancer detection by exploiting the exchange properties of hydroxyl protons [1, 2]. Several MRI methods have been shown to have the ability to detect glucose signal in vivo including the conventional continuous wave chemical exchange saturation transfer (cw-CEST), R2 relaxation enhancement [3, 4] and chemical exchange sensitive spin lock (CESL) [5, 6]. When D-glucose enters the tumor, it diffuses into the extravascular extracellular space (EES) due to disruption of the blood brain barrier (BBB). Unlike gadolinium-based contrast agents, glucose can also be actively taken up in the cells via facilitated transport and metabolized. In this study, a new on-resonance variable delay multi-pulse (onVDMP) method, which can be optimized to detect fast-exchanging protons such as the hydroxyl protons in D-glucose, was applied to study glucoCEST on a mouse with a 9L tumor. Dynamic glucose enhanced (DGE) images [7] were acquired using both the new technique and cw-CEST for comparison. Animal preparation: 9L glioma cells were implanted (0.05×105 cells/μl) by stereotaxic injection into the right caudate/putamen of female SCID mice. Imaging was performed on day 7 (cw-CEST) and 8 (onVDMP) post implantation. CEST imaging: Mice were anesthetized using isoflurane and positioned in an 11.7T horizontal bore Bruker Biospec scanner. For cw-CEST, DGE images were acquired at a single frequency of 1.2 ppm and saturation was achieved by a single magnetization transfer (MT) pre-pulse (3s, B1=1.6 μT). For onVDMP, 32 binomial pulses with 20 ms mixing time between pulse pairs were applied. The binomial pulses consisted of two 1.5 ms 46.8 μT pulses of opposite phase (Fig. 1A). Since a high saturation power is applied for a brief period in onVDMP, it is more sensitive to fast-exchanging protons. DGE Images were acquired using a Rapid Acquisition with Relaxation Enhancement (RARE) sequence, TR/TE=5.0 s/3.8 ms. The time for acquiring each dynamic scan was 10 s for both methods. 0.15 mL 50% w/w glucose was given over 60 s through the tail vein during the DGE scans. Data analysis: dynamic difference images were generated by taking the difference between each dynamic image and the average of all pre-injection images. Contrast to noise ratio (CNR) was calculated from the difference images:$$CNR(t) = \frac{S(t)_{tumor}-S(t)_{brain}}{\sqrt{2}SD(s_{i+1}-s_{i})},$$ in which the standard deviation (SD) is a measure of variation in noise over two consecutive acquisitions, $$$s_{i} $$$ and $$$s_{i+1}.$$$Figures 1a and 1b illustrate the onVDMP and cw-CEST sequences. Bloch simulations were performed to show CEST signal as a function of exchange rates using the MRI parameters described in the Methods section. Both methods have similar sensitivities for protons with exchange rate around 1kHz, such as some hydroxyl groups in glucose under physiological conditions. However for protons with higher exchange rates (> 1kHz), the sensitivity of cw-CEST decreases rapidly while that for onVDMP increases. The dynamic difference images from one mouse imaged on two consecutive days using the two methods are shown in Figure 2. It can be seen that both methods highlight the tumor and blood vessels upon glucose injection. When comparing the dynamic CNR (Figure 3) it can be seen the onVDMP method has in general 1.5 times higher CNR than the cw-CEST. There are 5 hydroxyl protons in each glucose molecule and their exchange rates range from 1 kHz to 3 kHz. Since the cw-CEST images were acquired at 1.2 ppm, only 3 out of 5 hydroxyl protons resonate around that frequency.[8] On the other hand, since onVDMP is not frequency selective, all fast exchanging protons contribute to the observed signal explaining the large gain in CNR. The onVDMP method is similar to spin-lock approaches and the on-resonance WALTZ-16 method [9] in the way that it is not frequency selective, but provides much higher sensitivity by detecting all fast-exchanging protons simultaneously.We compared the glucoCEST signal change upon venous glucose injection using cw-CEST and a newly developed onVDMP method. It has potential for widespread application for monitoring CEST contrast agents and drug delivery for compounds with fast-exchanging protons.