Development of a hyperpolarized ¹³C MRI pulse sequence for acquiring high-resolution 3D images after a single excitation.Imaging tumour responses to treatment through changes in metabolism can be advantageous as the morphological changes observed using traditional imaging methods can be slow to show evidence of a treatment response. Imaging of ¹³C-labelled cell substrates has become a promising technique for tumor treatment monitoring with the development of DNP in the past few years ^[1]. However, the hyperpolarization is transient, limiting the imaging time and number of excitation pulses in the experiment. We describe here a single-shot 3D imaging sequence to image hyperpolarized ¹³C-labeled substrates at high resolution, both spatially and temporally.The sequence is shown in Figure 1. Following each SpSp excitation pulse, the pulse sequence acquires a stack-of-spirals such that each kx-ky plane, in 3D k-space, is acquired with a spiral-out trajectory and the blipping gradient train encodes the spatial information in the z-direction. The spirals are interleaved to reduce the impact of system imperfections. The two pairs of Hyperbolic-Secant adiabatic inversion pulses form two spin echoes, which are acquired by the two central spirals, enhancing the overall SNR while reducing polarization depletion due to imperfect refocusing pulses. The design of the spiral trains can be varied depending on the requirements of the application: as displayed in Figure 1, acquiring larger matrices at the center of k-space and smaller ones at the periphery yields a 3D k-space with a near-spherical stack-of-spirals. Alternatively an identical matrix size for all kx-ky planes can be used, resulting in a cylindrical stack-of-spirals. Different designs result in different 3D point spread functions; choosing between them can be used to balance the in-plane resolution against the through-plane resolution.Experiments were performed on a 7T scanner with 3 female C57BL/6J mice bearing EL4 lymphoma tumours. Imaging commenced just before intravenous injection of hyperpolarized [1-¹³C]pyruvate, with a FOV of 4x4x2 cm³ and a nominal resolution of 1.25x1.25x2.5 mm³. The 1/3/5/7 (or, identically, 8/6/4/2) spirals ^[2], as shown in Figure 1, encoded matrix sizes of 4x4, 16x16, 32x32, 8x8, respectively. The spiral lengths, in the same order, were 0.656, 3.240, 8.580, and 1.344 ms. The SpSp pulse had a bandwidth of 350 Hz, a stop band of 1295 Hz, and a total duration of 10.056 ms. The flip angle was 15⁰ on pyruvate and 90⁰ on lactate, following a saturation-recovery scheme ^[3]. The TE1 and TE2, as indexed in Figure 1, were 50.6 ms and 110.504 ms respectively. The SpSp pulse was targeted alternately at the pyruvate and lactate resonances, giving a temporal resolution of 2 s per metabolite (TR = 1 s), while the total acquisition window was 60 s. Phase correction ^[4] was performed before the reconstruction process to remove the gross phase inconsistencies between each acquired spiral.The time courses of the signals from pyruvate and lactate are shown for all 3 mice in Figure 2, and were calculated by summing the signal from all the pixels in the 4^th slice. Lactate signal intensity was tightly correlated with the pyruvate signal strength, because the accumulation of lactate signal between excitations was prevented by the use of a 90⁰ flip angle pulse on the lactate resonance. The pyruvate and lactate images from the 4^th slice, overlaid on the proton Fast Spin Echo (FSE) image at the same position, are shown in Figure 3 for one mouse, between 0 s and 29 s. For the same mouse, the pyruvate and lactate signals are displayed in Figure 4 for all 8 slices at 6 s and 7 s respectively, overlaid on the proton FSE images. Tumour heterogeneity can be observed from the inhomogeneous distribution of the pyruvate and lactate signals.The proposed pulse sequence is, to the authors’ best knowledge, the first single-shot 3D sequence for hyperpolarized ¹³C MRI. The single-shot scheme can dramatically reduce the required number of excitations compared to earlier 3D techniques. Compared to 2D imaging methods, the sequence is beneficial in terms of both the reduced number of excitations and higher SNR. It has been shown to be capable of imaging exchange of ¹³C label between injected [1-¹³C]pyruvate and endogenous lactate in vivo at high spatial and temporal resolutions. Variations in the design of the spiral train allow different trade-offs between the in-plane and through-plane resolutions. The total duration of the sequence is much smaller than the TR used, leaving room for higher temporal resolution if required.