High-resolution hyperpolarized in vivo metabolic 13C spectroscopy at low magnetic field (48.7mT) following murine tail-vein injection.

High-resolution 13C NMR spectroscopy of hyperpolarized succinate-1-13C-2,3-d2 is reported in vitro and in vivo using a clinical-scale, biplanar (80cm-gap) 48.7mT permanent magnet with a high homogeneity magnetic field. Non-localized 13C NMR spectra were recorded at 0.52MHz resonance frequency over the torso of a tumor-bearing mouse every 2s. Hyperpolarized 13C NMR signals with linewidths of ∼3Hz (corresponding to ∼6ppm) were recorded in vitro (2mL in a syringe) and in vivo (over a mouse torso). Comparison of the full width at half maximum (FWHM) for 13C NMR spectra acquired at 48.7mT and at 4.7T in a small-animal MRI scanner demonstrates a factor of ∼12 improvement for the 13C resonance linewidth attainable at 48.7mT compared to that at 4.7T in vitro. 13C hyperpolarized succinate-1-13C resonance linewidths in vivo are at least one order of magnitude narrower at 48.7mT compared to those observed in high-field (≥3T) studies employing HP contrast agents. The demonstrated high-resolution 13C in vivo spectroscopy could be useful for high-sensitivity spectroscopic studies involving monitoring HP agent uptake or detecting metabolism using HP contrast agents with sufficiently large 13C chemical shift differences.

Open-Source Automated Parahydrogen Hyperpolarizer for Molecular Imaging Using (13)C Metabolic Contrast Agents.

An open-source hyperpolarizer producing (13)C hyperpolarized contrast agents using parahydrogen induced polarization (PHIP) for biomedical and other applications is presented. This PHIP hyperpolarizer utilizes an Arduino microcontroller in conjunction with a readily modified graphical user interface written in the open-source processing software environment to completely control the PHIP hyperpolarization process including remotely triggering an NMR spectrometer for efficient production of payloads of hyperpolarized contrast agent and in situ quality assurance of the produced hyperpolarization. Key advantages of this hyperpolarizer include: (i) use of open-source software and hardware seamlessly allowing for replication and further improvement as well as readily customizable integration with other NMR spectrometers or MRI scanners (i.e., this is a multiplatform design), (ii) relatively low cost and robustness, and (iii) in situ detection capability and complete automation. The device performance is demonstrated by production of a dose (∼2-3 mL) of hyperpolarized (13)C-succinate with %P13C ∼ 28% and 30 mM concentration and (13)C-phospholactate at %P13C ∼ 15% and 25 mM concentration in aqueous medium. These contrast agents are used for ultrafast molecular imaging and spectroscopy at 4.7 and 0.0475 T. In particular, the conversion of hyperpolarized (13)C-phospholactate to (13)C-lactate in vivo is used here to demonstrate the feasibility of ultrafast multislice (13)C MRI after tail vein injection of hyperpolarized (13)C-phospholactate in mice.

Aqueous NMR Signal Enhancement by Reversible Exchange in a Single Step Using Water-Soluble Catalysts.

Two synthetic strategies are investigated for the preparation of water-soluble iridium-based catalysts for NMR signal amplification by reversible exchange (SABRE). In one approach, PEGylation of a variant N-heterocyclic carbene provided a novel catalyst with excellent water solubility. However, while SABRE-active in ethanol solutions, the catalyst lost activity in >50% water. In a second approach, synthesis of a novel di-iridium complex precursor where the cyclooctadiene (COD) rings have been replaced by CODDA (1,2-dihydroxy-3,7-cyclooctadiene) leads to the creation of a catalyst [IrCl(CODDA)IMes] that can be dissolved and activated in water-enabling aqueous SABRE in a single step, without need for either an organic cosolvent or solvent removal followed by aqueous reconstitution. The potential utility of the CODDA catalyst for aqueous SABRE is demonstrated with the ∼(-)32-fold enhancement of 1H signals of pyridine in water with only 1 atm of parahydrogen.

Hyperpolarization of "Neat" Liquids by NMR Signal Amplification by Reversible Exchange.

We report NMR Signal Amplification by Reversible Exchange (SABRE) hyperpolarization of the rare isotopes in "neat" liquids, each composed only of an otherwise pure target compound with isotopic natural abundance (n.a.) and millimolar concentrations of dissolved catalyst. Pyridine (Py) or Py derivatives are studied at 0.4% isotopic natural abundance ¹5N, deuterated, ¹5N enriched, and in various combinations using the SABRE-SHEATH variant (microTesla magnetic fields to permit direct ¹5N polarization from parahydrogen via reversible binding and exchange with an Ir catalyst). We find that the dilute n.a. ¹5N spin bath in Py still channels spin order from parahydrogen to dilute ¹5N spins, without polarization losses due to the presence of ¹4N or ²H. We demonstrate P(15N) ≈ 1% (a gain of 2900 fold relative to thermal polarization at 9.4 T) at high substrate concentrations. This fundamental finding has a significant practical benefit for screening potentially hyperpolarizable contrast agents without labeling. The capability of screening at n.a. level of ¹5N is demonstrated on examples of mono- and dimethyl-substituted Py (picolines and lutidines previously identified as promising pH sensors), showing that the presence of a methyl group in the ortho position significantly decreases SABRE hyperpolarization.

15N Hyperpolarization by Reversible Exchange Using SABRE-SHEATH.

NMR signal amplification by reversible exchange (SABRE) is a NMR hyperpolarization technique that enables nuclear spin polarization enhancement of molecules via concurrent chemical exchange of a target substrate and parahydrogen (the source of spin order) on an iridium catalyst. Recently, we demonstrated that conducting SABRE in microtesla fields provided by a magnetic shield enables up to 10% 15N-polarization (Theis, T.; et al. J. Am. Chem. Soc.2015, 137, 1404). Hyperpolarization on 15N (and heteronuclei in general) may be advantageous because of the long-lived nature of the hyperpolarization on 15N relative to the short-lived hyperpolarization of protons conventionally hyperpolarized by SABRE, in addition to wider chemical shift dispersion and absence of background signal. Here we show that these unprecedented polarization levels enable 15N magnetic resonance imaging. We also present a theoretical model for the hyperpolarization transfer to heteronuclei, and detail key parameters that should be optimized for efficient 15N-hyperpolarization. The effects of parahydrogen pressure, flow rate, sample temperature, catalyst-to-substrate ratio, relaxation time (T1), and reversible oxygen quenching are studied on a test system of 15N-pyridine in methanol-d4. Moreover, we demonstrate the first proof-of-principle 13C-hyperpolarization using this method. This simple hyperpolarization scheme only requires access to parahydrogen and a magnetic shield, and it provides large enough signal gains to enable one of the first 15N images (2 × 2 mm2 resolution). Importantly, this method enables hyperpolarization of molecular sites with NMR T1 relaxation times suitable for biomedical imaging and spectroscopy.

Microtesla SABRE enables 10% nitrogen-15 nuclear spin polarization.

Parahydrogen is demonstrated to efficiently transfer its nuclear spin hyperpolarization to nitrogen-15 in pyridine and nicotinamide (vitamin B(3) amide) by conducting "signal amplification by reversible exchange" (SABRE) at microtesla fields within a magnetic shield. Following transfer of the sample from the magnetic shield chamber to a conventional NMR spectrometer, the (15)N NMR signals for these molecules are enhanced by ∼30,000- and ∼20,000-fold at 9.4 T, corresponding to ∼10% and ∼7% nuclear spin polarization, respectively. This method, dubbed "SABRE in shield enables alignment transfer to heteronuclei" or "SABRE-SHEATH", promises to be a simple, cost-effective way to hyperpolarize heteronuclei. It may be particularly useful for in vivo applications because of longer hyperpolarization lifetimes, lack of background signal, and facile chemical-shift discrimination of different species.

Propane-d6 Heterogeneously Hyperpolarized by Parahydrogen.

Long-lived spin states of hyperpolarized propane-d6 gas were demonstrated following pairwise addition of parahydrogen gas to propene-d6 using heterogeneous parahydrogen-induced polarization (HET-PHIP). Hyperpolarized molecules were synthesized using Rh/TiO2 solid catalyst with 1.6 nm Rh nanoparticles. Hyperpolarized (PH ∼ 1%) propane-d6 was detected at high magnetic field (9.4 T) spectroscopically and by high-resolution 3D gradient-echo MRI (4.7 T) as the gas flowed through the radiofrequency coil with a spatial and temporal resolution of 0.5 × 0.5 × 0.5 mm3 and 17.7 s, respectively. Stopped-flow hyperpolarized propane-d6 gas was also detected at 0.0475 T with an observed nuclear spin polarization of PH ∼ 0.1% and a relatively long lifetime with T1,eff = 6.0 ± 0.3 s. Importantly, it was shown that the hyperpolarized protons of the deuterated product obtained via pairwise parahydrogen addition could be detected directly at low magnetic field. Importantly, the relatively long low-field T1,eff of HP propane-d6 gas is not susceptible to paramagnetic impurities as tested by exposure to ∼0.2 atm oxygen. This long lifetime and nontoxic nature of propane gas could be useful for bioimaging applications including potentially pulmonary low-field MRI. The feasibility of high-resolution low-field 2D gradient-echo MRI was demonstrated with 0.88 × 0.88 mm2 spatial and ∼0.7 s temporal resolution, respectively, at 0.0475 T.

In situ and ex situ low-field NMR spectroscopy and MRI endowed by SABRE hyperpolarization.

By using 5.75 and 47.5 mT nuclear magnetic resonance (NMR) spectroscopy, up to 10(5)-fold sensitivity enhancement through signal amplification by reversible exchange (SABRE) was enabled, and subsecond temporal resolution was used to monitor an exchange reaction that resulted in the buildup and decay of hyperpolarized species after parahydrogen bubbling. We demonstrated the high-resolution low-field proton magnetic resonance imaging (MRI) of pyridine in a 47.5 mT magnetic field endowed by SABRE. Molecular imaging (i.e. imaging of dilute hyperpolarized substances rather than the bulk medium) was conducted in two regimes: in situ real-time MRI of the reaction mixture (in which pyridine was hyperpolarized), and ex situ MRI (in which hyperpolarization decays) of the liquid hyperpolarized product. Low-field (milli-Tesla range, e.g. 5.75 and 47.5 mT used in this study) parahydrogen-enhanced NMR and MRI, which are free from the limitations of high-field magnetic resonance (including susceptibility-induced gradients of the static magnetic field at phase interfaces), potentially enables new imaging applications as well as differentiation of hyperpolarized chemical species on demand by exploiting spin manipulations with static and alternating magnetic fields.

Irreversible catalyst activation enables hyperpolarization and water solubility for NMR signal amplification by reversible exchange.

Activation of a catalyst [IrCl(COD)(IMes)] (IMes = 1,3-bis(2,4,6-trimethylphenyl)imidazol-2-ylidene; COD = cyclooctadiene)] for signal amplification by reversible exchange (SABRE) was monitored by in situ hyperpolarized proton NMR at 9.4 T. During the catalyst-activation process, the COD moiety undergoes hydrogenation that leads to its complete removal from the Ir complex. A transient hydride intermediate of the catalyst is observed via its hyperpolarized signatures, which could not be detected using conventional nonhyperpolarized solution NMR. SABRE enhancement of the pyridine substrate can be fully rendered only after removal of the COD moiety; failure to properly activate the catalyst in the presence of sufficient substrate can lead to irreversible deactivation consistent with oligomerization of the catalyst molecules. Following catalyst activation, results from selective RF-saturation studies support the hypothesis that substrate polarization at high field arises from nuclear cross-relaxation with hyperpolarized (1)H spins of the hydride/orthohydrogen spin bath. Importantly, the chemical changes that accompanied the catalyst's full activation were also found to endow the catalyst with water solubility, here used to demonstrate SABRE hyperpolarization of nicotinamide in water without the need for any organic cosolvent--paving the way to various biomedical applications of SABRE hyperpolarization methods.

Long-lived spin States for low-field hyperpolarized gas MRI.

Parahydrogen induced polarization was employed to prepare a relatively long-lived correlated nuclear spin state between methylene and methyl protons in propane gas. Conventionally, such states are converted into a strong NMR signal enhancement by transferring the reaction product to a high magnetic field in an adiabatic longitudinal transport after dissociation engenders net alignment (ALTADENA) experiment. However, the relaxation time T1 of ∼0.6 s of the resulting hyperpolarized propane is too short for potential biomedical applications. The presented alternative approach employs low-field MRI to preserve the initial correlated state with a much longer decay time TLLSS =(4.7±0.5) s. While the direct detection at low-magnetic fields (e.g. 0.0475 T) is challenging, we demonstrate here that spin-lock induced crossing (SLIC) at this low magnetic field transforms the long-lived correlated state into an observable nuclear magnetization suitable for MRI with sub-millimeter and sub-second spatial and temporal resolution, respectively. Propane is a non-toxic gas, and therefore, these results potentially enable low-cost high-resolution high-speed MRI of gases for functional imaging of lungs and other applications.

Sodium 3D COncentration MApping (COMA 3D) using (23)Na and proton MRI.

Functional changes of sodium 3D MRI signals were converted into millimolar concentration changes using an open-source fully automated MATLAB toolbox. These concentration changes are visualized via 3D sodium concentration maps, and they are overlaid over conventional 3D proton images to provide high-resolution co-registration for easy correlation of functional changes to anatomical regions. Nearly 5000/h concentration maps were generated on a personal computer (ca. 2012) using 21.1T 3D sodium MRI brain images of live rats with spatial resolution of 0.8×0.8×0.8 mm(3) and imaging matrices of 60×60×60. The produced concentration maps allowed for non-invasive quantitative measurement of in vivo sodium concentration in the normal rat brain as a functional response to migraine-like conditions. The presented work can also be applied to sodium-associated changes in migraine, cancer, and other metabolic abnormalities that can be sensed by molecular imaging. The MATLAB toolbox allows for automated image analysis of the 3D images acquired on the Bruker platform and can be extended to other imaging platforms. The resulting images are presented in a form of series of 2D slices in all three dimensions in native MATLAB and PDF formats. The following is provided: (a) MATLAB source code for image processing, (b) the detailed processing procedures, (c) description of the code and all sub-routines, (d) example data sets of initial and processed data. The toolbox can be downloaded at: http://www.vuiis.vanderbilt.edu/~truongm/COMA3D/.

High-resolution 3D proton MRI of hyperpolarized gas enabled by parahydrogen and Rh/TiO2 heterogeneous catalyst.

Several supported metal catalysts were synthesized, characterized, and tested in heterogeneous hydrogenation of propene with parahydrogen to maximize nuclear spin hyperpolarization of propane gas using parahydrogen induced polarization (PHIP). The Rh/TiO2 catalyst with a metal particle size of 1.6 nm was found to be the most active and effective in the pairwise hydrogen addition and robust, demonstrating reproducible results with multiple hydrogenation experiments and stability for ≥1.5 years. 3D (1) H magnetic resonance imaging (MRI) of 1 % hyperpolarized flowing gas with microscale spatial resolution (625×625×625 µm(3) ) and large imaging matrix (128×128×32) was demonstrated by using a preclinical 4.7 T scanner and 17.4 s imaging scan time.

Sub-second proton imaging of 13C hyperpolarized contrast agents in water.

Indirect proton detection of (13)C hyperpolarized contrast agents potentially enables greater sensitivity. Presented here is a study of sub-second projection imaging of hyperpolarized (13)C contrast agent addressing the obstacle posed by water suppression for indirect detection in vivo. Sodium acetate phantoms were used to develop and test water suppression and sub-second imaging with frequency-selective RF pulses using spectroscopic and imaging indirect proton detection. A 9.8 mm aqueous solution of (13)C PHIP hyperpolarized 2-hydroxyethyl-(13)C-propionate-d2,3,3 (HEP),

~25% was used for demonstration of indirect proton sub-second imaging detection. Balanced 2D FSSFP (fast steady-state free precession) allowed the recording of proton images with a field of view of 64 × 64 mm(2) and spatial resolution 2 × 2 mm(2) with total acquisition time of less than 0.2 s. In thermally polarized sodium 1-(13)C-acetate, (13) C to (1)H polarization transfer efficiency of 45.1% of the theoretically predicted values was observed in imaging detection corresponding to an 11-fold overall sensitivity improvement compared with direct (13)C FSSFP imaging. (13)C to (1)H polarization transfer efficiency of 27% was observed in imaging detection, corresponding to a 3.25-fold sensitivity improvement compared with direct (13)C FSSFP imaging with hyperpolarized HEP. The range of potential applications and limitations of this sub-second and ultra-sensitive imaging approach are discussed.

Low-field MRI can be more sensitive than high-field MRI.

MRI signal-to-noise ratio (SNR) is the key factor for image quality. Conventionally, SNR is proportional to nuclear spin polarization, which scales linearly with magnetic field strength. Yet ever-stronger magnets present numerous technical and financial limitations. Low-field MRI can mitigate these constraints with equivalent SNR from non-equilibrium 'hyperpolarization' schemes, which increase polarization by orders of magnitude independently of the magnetic field. Here, theory and experimental validation demonstrate that combination of field independent polarization (e.g. hyperpolarization) with frequency optimized MRI detection coils (i.e. multi-turn coils using the maximum allowed conductor length) results in low-field MRI sensitivity approaching and even rivaling that of high-field MRI. Four read-out frequencies were tested using samples with identical numbers of (1)H and (13)C spins. Experimental SNRs at 0.0475T were ∼40% of those obtained at 4.7T. Conservatively, theoretical SNRs at 0.0475T 1.13-fold higher than those at 4.7T were possible despite an ∼100-fold lower detection frequency, indicating feasibility of high-sensitivity MRI without technically challenging, expensive high-field magnets. The data at 4.7T and 0.0475T was obtained from different spectrometers with different RF probes. The SNR comparison between the two field strengths accounted for many differences in parameters such as system noise figures and variations in the probe detection coils including Q factors and coil diameters.