Decoupling of Magnetic Resonance Signals in Time and Frequency Domain

Nuclear magnetic resonance (NMR) was discovered in the first half of the 20th century. Today, neither analytical chemistry without NMR spectroscopy nor medical diagnostics without magnetic resonance imaging (MRI) could be imagined. A magnetic resonance signal decays with a time constant T2, the transverse relaxation time. This parameter contains a great deal of information about structure and dynamics of the molecule, where the observed nuclear spin is located. A method to measure T2 is the so-called spin echo, discovered by Erwin Hahn in 1950. Usually, the spin echo can generate an exponential decay of the signal and T2 can be extracted. But if the indirect spin-spin interaction, the J -coupling, connects nuclear spins of the same isotope, the echo signal is modulated. The present work discusses novel methods to quench these echo modulations. This enables one, on the one hand, to measure the transverse relaxation in homonuclear spin systems. On the other hand, this decoupling technique can simplify NMR spectra by making splittings due to J-couplings disappear. These two processes can be seen to be equivalent, since the Fourier transform of a oscillating time signal corresponds to a line splitting of the frequency signal. To measure T2’s of protons (the nuclei of the most abundant hydrogen isotope 1H) or molecules isotopically enriched in carbon-13, we use a “train of echo pulses” with a moderate amplitude of the irradiated radio-frequency. To decouple various nuclear spins in a 1H NMR spectrum, we use a selective radio-frequency irradiation. These experiments are performed as Fourier transform spectroscopy, introduced by Ernst and Anderson in 1966. Based on the same pulse sequence, we measure so-called “spin tickling” experiments, described by Freeman and Anderson in 1962. All lines in the spectrum split that share a common energy-level with the irradiated resonance. “Tickling” and decoupling are complementary and belong both to the category of double resonance NMR experiments.

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

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

Daniele Mammoli

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.

EPFL2016

Chemical exchange in proteins

Mariachiara Verde

Nuclear Magnetic Resonance (NMR) spectroscopy offers many ways to investigate dynamic properties of molecules. A wide variety of experimental techniques can probe molecular dynamics on time scales that range from 10-12 to 103 seconds. Many lines of evidence point to the existence of large scale dynamics on milli- to microsecond time scale that are essential to molecular function. Those conformational motions can give rise to chemical exchange effects. So far in the literature two methods have been mainly used to study such processes: CPMG echo trains and R1ρ spin-lock relaxation experiments. The former is suited to investigate processes on the millisecond time scale, whereas R1ρ can probe processes ranging from milliseconds down to ca. microseconds. CPMG and R1ρ studies of the relaxation rates of single quantum (SQ) coherences can be complemented by multiple quantum coherence (MQ) coherences experiments which can provide information whether two (or more) spins are affected simultaneously by chemical exchange. The goal of this thesis is to characterize chemical exchange in proteins by new (Heteronuclear Double Resonance, HDR) and existing (relaxation compensated CPMG) methods. The presence of chemical exchange has been identified in APO-rMUP using the classical CPMG experiment on single quantum 15N magnetization, whereas in HOLO-rMUP no presence of chemical exchange has been found. Most of the thesis is dedicated to the analysis and the application of a new method, HDR, which is designed to determine the cross-relaxation rates between MQ operators. In fact, in heteronuclear systems, correlated chemical exchange contributes to cross-relaxation between MQ coherences 2IxSx and 2IySy. This method, based on MLEV-32 and WALTZ-16 used in double resonance mode, is designed to effectively preserve all relevant MQ coherences simultaneously so that the interconversion between MQ operators can only arise through cross-relaxation. This is an essential requirement if we want to measure, for example, small cross-relaxation rates between MQ operators. We have treated the effects of coherent evolution during the HDR sequences by numerical simulations and Average Hamiltonian Theory (AHT) showing that, under most conditions, the dynamics of MQ coherences is not affected by coherent evolution. This result is proved experimentally on the 15N-1H pair of selectively deuterated tBoc-glycine. The effect of relaxation during HDR sequences leads to an effective relaxation superoperator which is explored by numerical simulations and predicted by Average Liouvillian Theory (ALT). Finally the HDR MLEV-32 sequence is applied to amide 1H-15N pairs in ubiquitin and KIX proteins. In the case of ubiquitin the HDR MLEV-32 experiments and the numerical simulations run with parameters obtained from the literature are in good agreement, suggesting that HDR sequences could be used as a tool to obtain information about the dynamics of the system.

EPFL2009