Long-lived nuclear spin states of magnetically equivalent protons by means of dissolution dynamic nuclear polarization

Nuclear Magnetic Resonance (NMR) is one of the most versatile techniques since it enables the characterization of solid, liquid and gaseous systems in a plethora of in-vitro and in-vivo experiments. Despite its multidisciplinary scope, it still suffers from two drawbacks, namely the intrinsic low sensitivity and the fast return to equilibrium. Dissolution Dynamic Nuclear Polarization (D-DNP) is a powerful scheme to enhance the signals of nuclear spins by up to five orders of magnitude and it is at the forefront of current research in NMR. The main detriment is that the great efforts spent in producing the hard-earned hyperpolarized signals fade because of rapid longitudinal relaxation with a time constant T1. Long-Lived States (LLS) are states with lifetimes that may be much longer than T1. In a two-spin system, they can be defined as a T/S Imbalance (TSI) between the average population of the three triplet (T) states on the one hand and the population of the singlet (S) state on the other. So far, para-hydrogen is the only molecule routinely used as a source of long-lived hyperpolarization. In recent years, it has been shown that it is possible to create a TSI in magnetically in-equivalent spins, by exploiting DNP to lower the spin temperature well below the Zeeman splitting. The present thesis is devoted to combine D-DNP with LLS in the perspective of preparing enhanced and long-lasting NMR signals in systems comprising magnetically equivalent protons. We proved that that is possible to induce the TSI via D-DNP in partly deuterated ethanol and in fumaric acid. In ethanol the TSI has been revealed by 1H-13C cross-relaxation that leads to population differences across observable transitions. In fumarate, we resorted to a symmetry-breaking addition of D2O catalysed by fumarase which makes the TSI observable on the two magnetically in-equivalent protons of malate. In principle, our strategy should allow one to prepare either an excess or a deficiency of para-water, which we like to refer to as âforbidden fruitsâ of spectroscopy. Water is challenging since its TSI may suffer from relaxation due to fast proton exchange and to spin-rotation. We assessed conditions suitable to slow down proton exchange by dilution in aprotic solvents, yielding lifetimes of water as a molecular entity on a timescale compatible with D-DNP experiments. We designed an arrangement of coaxial tubes to measure T1 of water vapour at known pressures and temperatures. The experimental T1 are rather small (i.e., tens of ms) and in fair agreement with predictions if spin-rotation is the dominant relaxation mechanism. Empirical evidence of the survival of the hyperpolarized signal of water after dissolution in our lab suggests that the exchange of water molecules between the gaseous and liquid phases is either inefficient or slow in most of cases we studied. I report preliminary results of the application of our D-DNP scheme to induce a TSI in water. Considering the complexity of the project, further investigation is needed to unravel and optimize recent observations in view of identifying an unambiguous fingerprint of dilute para-water.

Methods for Molecular Magnetic Resonance Imaging and Magnetic Resonance Spectroscopy using Hyperpolarized Nuclei

Riccardo Balzan

Since the introduction 10 years ago of the dissolution method, Dynamic Nuclear Polarization (DNP) became a widely applied and powerful technique to enhance nuclear magnetic resonance (NMR) signals of low naturally abundant, insensitive nuclear spins for analytical chemistry and biomedical research. The aim of DNP is to obtain a very high degree of polarization on the nuclei of interest in cryogenic conditions (i.e. 1 K ), a hundred or more times higher than the thermal polarization. Then, trough a fast dissolution process the molecules hosting the nuclear spins are mixed in a room temperature solution and, since their polarization is conserved if their relaxation times are long enough, they can be used for in vitro or in vivo experiments with a SNR enhanced by 104 times or more. Although the DNP technique was established about 60 years ago, the hardware and cryogenic equipment needed to perform DNP and the subsequent dissolution, still are a technological challenge. In this thesis we will cover different DNP aspects, ranging from hardware installation, software development, solid state measurement, in vitro and in vivo experiments and a possible application of DNP to MRI studies of granular materials. A new DNP cryostat was developed and tested to determine its cryogenic performances and then optimized for fast cooldown, helium holdtime and overall minimal operational consumption. We will show the characteristics of the cryostat and the performances on standard operations like cooling and DNP at 1 K . Another important aspect the development tackled was automation. The management of the system was condensed in a single electronic box capabile to handle the interface with all the cryostat instruments. This box is driven by a single common USB port trough a custom made software interface we developed. Although a few manual operations are still needed, we achieved a high degree of automation. The solid state polarization enhancement ε is defined as the ratio between the DNP enhanced and the thermal polarization signals. It can be determined by measuring the DNP enhanced signal after the system stabilizes in the polarized state (i.e. after about 5 buildup time) and thermal polarization signal after the spins have fully relaxed to equilibrium (i.e. after about 5T1). As a first application of the new system, we present a method that exploits the behavior of steady states magnetization produced with trains of evenly spaced pulses, at constant flip angle. This method allows to precisely determine the thermally polarized signal of a sample with known T1 and a given flip angle in a small fraction (as low as half of a T1 or less depending on the requested accuracy) of the time needed in the usual "wait 5 T1 and pulse" scheme. The main drawback of the dissolution DNP is that the large polarization achieved will ineluctably relax towards its thermal equilibrium value in a few times T1, that for typical 13C labeled molecules is shorter than a minute. On the other hand some nuclei as 6Li and quaternary 15N show a very long T1 on the order of few minutes in biological conditions, allowing measurements as long as several minutes. 6Li is very sensitive to contrast agents such as Gd-doped contrast agents, O2 and de-oxygenated hemoglobin. We studied the effect of the oxygenation of human and rat blood on the 6Li T1, showing clear changes between oxygenated and de-oxygenated samples. Thus we showed that 6Li could be used to map blood oxygenation levels in vitro, and suggest possible in vivo applications, even in brain due to its capability to easily pass the blood brain barrier. 15N labeled choline has a very long 15N T1 and can be used to probe rate of transport or metabolism in brain. Although it has a long T1, the spectral dispersion between the precursor, choline, and its metabolites is very small. The spectral dispersion is much larger for 1 H spins and thus being able to partially transfer repeatedly the 15N to 1H before detection would greatly increase the spectral separation between metabolites while still being able to measure the time course of the metabolic process, which is not the case with a full transfer like with INEPT. We demonstrate that a Hartmann–Hahn polarization transfer can be used to partially transfer the hyperpolarization of 15N to surrounding 1H before detection on 1H. This method can then be applied to in vivo DNP experiments. Even with a very high signal it is very difficult to quantify metabolites that are very close in the NMR spectrum, compared to the actual in vivo linewidth. To improve the spectral resolution it is worth performing the experiments in high magnetic fields. We show the first in vivo rat muscle DNP measurement of acetate at 14.1 T , to resolve e.g. the glutamate peak from acetate substrate. Finally we discuss a segmentation algorithm used to precisely reconstruct granular samples composed of up to 104 spheres. We used a standard Gradient Echo imaging sequence on a Siemens 7 T MRI human scanner to obtain high resolution images of a phantom composed of a cylinder filled with Cu2+ doped water and plastic spheres. Then the 3D images where segmented trough a thresholded Hugh transform method and the beads centers determined. The datasets were studied to extract structural and geometrical information.

EPFL2013

Collisional cross-section of water molecules in vapour studied by means of H-1 relaxation in NMR

Geoffrey Bodenhausen, Roberto Buratto, Estel Canet Martinez, Lothar Helm, Daniele Mammoli, Pascal Miéville

In gas phase, collisions that affect the rotational angular momentum lead to the return of the magnetization to its equilibrium (relaxation) in Nuclear Magnetic Resonance (NMR). To the best of our knowledge, the longitudinal relaxation rates R-1 = 1/T-1 of protons in H2O and HDO have never been measured in gas phase. We report R1 in gas phase in a field of 18.8 T, i.e., at a proton Larmor frequency v(0) = 800 MHz, at temperatures between 353 and 373 K and pressures between 9 and 101 kPa. By assuming that spin rotation is the dominant relaxation mechanism, we estimated the effective cross-section sigma(J) for the transfer of angular momentum due to H2O-H2O and HDO-D2O collisions. Our results allow one to test theoretical predictions of the intermolecular potential of water in gas phase.

Nature Publishing Group2016

Ultra high magnetic field for glial contribution into brain metabolism studied by MR spectroscopy and CEST methods for molecular imaging of glycogen

Elise Marie Catherine Vinckenbosch

Magnetic resonance at ultra-high field increases signal and spectral dispersion. In this thesis, I use those characteristics to investigate three different subjects in 13C spectroscopy, hyperpolarized methods and chemical exchange saturation transfer (CEST) used for molecular imaging. In the brain, metabolism is driven by glia-neuron interactions and is composed of at least two compartments with distinct TCA cycle kinetics. The first study examines cerebral metabolic adaptation after an induced glial TCA-cycle impairment using 1H and 13C spectroscopy. We treated rats with fluoroacetate, which specifically blocks glial aconitase and measured changes in neurochemical profile at euglycemic and hyperglycemic condi-tions by in vivo 1H MR spectroscopy. In addition, we evaluated the brain-compartmentalized metabolic adap-tation by following 13C-incorporation into brain amino acids by modern dynamic 13C MRS methods during infu-sion of [1,6-13C2]glucose. By following [1,6-13C2]glucose incorporation, we successfully observed an augmen-tation of pyruvate carboxylase and glutamine synthetase fluxes and a 20% decrease of glial TCA cycle flux. Neuronal metabolism was also affected. In the second study, we focused on hyperpolarized methods to increase acetate 13C polarization allowing us to follow the kinetics of its metabolic products in the TCA cycle. Indeed, infusion of hyperpolarized [1-13C]acetate, an astrocyte-specific precursor, and the in vivo detection of 2-oxo[5-13C]glutarate (2OG), a TCA intermediate, enables the direct analysis of glial Krebs cycle activity. We examined the effect of hyperpolar-ized acetate concentration on its cerebral metabolism and calculate the production rate of 2OG. A healthy group was compared to rodents treated with fluoroacetate. The conversion rates of acetate to 2OG were calculated and were found to depend on the substrate dose. We successfully estimated 2OG production rates. In partially inhibited TCA cycle conditions, the production rate of 2OG was reduced by a quarter. This level of reduction is agrees with the percentage of inhibition calculated in the first study. Finally, we investigated glycogen imaging in skeletal muscle using the CEST strategy. It has been demonstrat-ed in perfused liver that glycogen can be detected indirectly through the water signal using CEST. Glycogen detection is, due to its resonance close to water, strongly sensitive to spectral dispersion and improved by a higher static magnetic field. In contrast to liver, which contains ~300 mmol/g of glycogen and enjoys good glycogen specificity, myocellular content is also rich in creatine, which produces a spectral signal on the same order of amplitude as its glycogen content of ~80-30 mmol/g. In this study, we allowed glycogen to be de-tectable in vivo and distinguishable from the neighboring creatine at 14.1T. We propose to use creatine as an internal reference for muscular glycoCEST analysis. Scanning muscle after physical exercise, resulted in a 50% reduction of glycoCEST compared to muscle at resting state. This result supportis the specificity of the CEST method and is consistent with literature. This last study demonstrates for the first time that glycoCEST imag-ing is feasible in vivo.