New pulse sequences for T1− and T1/T2−contrast enhancing in NMR imaging

Improved pulse sequences DIFN (abbreviation of the words: DIFferentiation by N pulses), 90° − τ₁ − 180° − τ₁ − … 180° − τ_n, with optimised time intervals τ₁ for T₁ measurement and contrast enhancing in NMR imaging are presented. The pulse sequences DIFN have a better sensitivity to T₁ than the well-known pulse sequence SR. In contrast to the IR pulse sequence, the information given by the DIFN pulse sequence is more reliable, because the NMR signal does not change its sign. For a given time interval τ₀ ≤ (0.1 − 0.3) T₁′ the DIFN pulse sequences serve as T₁-filters. They pass the signal components with relatively short T₁ < T₁′ and suppress the components with relatively long T₁ < T₁′. The effects of the radiofrequency field inhomogeneity and inaccurate adjusting of pulse lengths are also considered. It is also proposed in this work to use the joint T₁T₂-contrast in NMR imaging obtained as a result of applying the DIFN pulse sequences in combination with the well-known Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence. The region of interest, where the contrast should be especially enhanced, is specified by the two times at which measurements are performed, which allow the amplitudes of pixels to reach some defined levels by spin-lattice and spin-spin relaxation.     

Fluorine-19 Solid-State NMR Magic-Angle-Turning Experiments Using Multiple-Pulse Homonuclear Decoupling

For compounds giving “crowded” 1-dimensional magic-angle-spinning spectra, information about the local atomic environment in the form of the chemical shift anisotropy (CSA) is sacrificed for high resolution of the less informative isotropic chemical shift. Magic-angle-turning (MAT) NMR pulse sequences preserve the CSA information by correlating it to the isotropic chemical shift in a 2-dimensional experiment. For low natural abundance nuclei such as ¹³C and ¹⁵N and under ¹H heteronuclear dipolar decoupling conditions, the dominant NMR interaction is the chemical shift. For abundant nuclei such as ¹H, ¹⁹F, and ³¹P, the homonuclear dipolar interaction becomes a significant contribution to the observed linewidth in both F₁ and F₂ dimensions. We incorporate MREV8 homonuclear multiple-pulse decoupling sequences into the MAT experiment to give a multiple-pulse MAT (MP-MAT) experiment in which the homonuclear dipolar interaction is suppressed while maintaining the chemical shift information. Extensive use of computer simulation using GAMMA has guided the pulse sequence development. In particular, we show how the MREV8 pulses can be incorporated into a quadrature-detected sequence such as MAT. The MP-MAT technique is demonstrated for a model two-site system containing a mixture of silver trifluoroacetate and calcium difluoride. The resolution in the isotropic evolution dimension is improved by faster sample spinning, shorter MREV8 cycle times in the evolution dimension, and modifications of the MAT component of the pulse sequence.     

Complete Dipolar Decoupling ofC and Its Use in Two-Dimensional Double-Quantum Solid-State NMR for Determining Polymer Conformations

A multiple-pulse technique for complete dipolar decoupling of directly bonded¹³C-labeled sites is described. It achieves significant spectral simplifications in a recently introduced two-dimensional double-quantum solid-state NMR experiment for determining torsion angles. Both homonuclear and heteronuclear dipolar couplings are removed by combining a¹³C multiple-pulse sequence with continuous-wave irradiation on the protons. The¹³C sequence has a fundamental 10-pulse cycle which is a significantly modified magic-sandwich-echo sequence. The crucial heteronuclear decoupling is achieved by breaking the 360° “inner” pulses in the magic sandwich into 90° pulses and spacing them by¹H 360° pulse lengths. Spectral artifacts typical of multiple-pulse sequences are eliminated by phase shifts between cycles. In contrast to many other multiple-pulse decoupling sequences, the long window in the cycle is the dwell time and can be longer than the inverse dipolar coupling, which makes the sequence practical for direct detection even with long pulse ring-down times. A modification of the sequence to scale the chemical shift and increase the effective spectral width is also presented. The 1D and double-quantum 2D experiments are demonstrated on polyethylene with 4%¹³C–¹³C spin pairs. The potential of this approach for distinguishing segmental conformations is illustrated by spectral simulations of the two-dimensional ridge patterns that correlate double-quantum and single-quantum chemical-shift anisotropies.     

High resolution heteronuclear correlation NMR spectroscopy between quadrupolar nuclei and protons in the solid state

A high resolution two-dimensional solid state NMR experiment is presented that correlates half-integer quadrupolar spins with protons. In this experiment the quadrupolar nuclei evolve during t1 under a split-t1, FAM-enhanced MQMAS pulse scheme. After each t1 period ending at the MQMAS echo position, single quantum magnetization is transferred, via a cross polarization process in the mixing time, from the quadrupolar nuclei to the protons. High-resolution proton signals are then detected in the t2 time domain during wPMLG5* homonuclear decoupling. The experiment has been demonstrated on a powder sample of sodium citrate and 23Na-1H 2D correlation spectra have been obtained. From the HETCOR spectra and the regular MQMAS spectrum, the three crystallographically inequivalent Na+ sites in the asymmetric unit were assigned. This MQMAS-wPMLG HETCOR pulse sequence can be used for spectral editing of half-integer quadrupolar nuclei coupled to protons.     

Simplifying DOSY spectra with selective TOCSY edited preparation

Diffusion-ordered NMR spectroscopy, while quite powerful, is limited by its inability to resolve signals that are severely overlapped in the proton spectrum. We present here a DOSY experiment that uses selective TOCSY as an editing/preparation period. With this method, well-resolved signals of the analytes are selectively excited and the magnetization subsequently transferred by isotropic mixing to resonances buried in the matrix background, which are then resolved by the ensuing DOSY sequence. Key to the success of our proposed method is the incorporation of a highly effective zero-quantum filter into the selective TOCSY preparation period, which prevents zero-quantum coherence from being carried into the DOSY part of the pulse sequence. Further improvement in spectral resolution can be obtained by expanding the proposed experiment into a 3D sequence and utilizing the homonuclear decoupling feature of the BASHD-TOCSY technique. Both pulse sequences were found to greatly simplify the DOSY spectrum of a 'dirty' sucrose/raffinose mixture, as the complex matrix background is no longer present to obscure or overlap with the signals of interests. Furthermore, complete resolution of the relevant signals was achieved with the 3D sequence.     

17O-decoupled 1H detection using a double-tuned coil

¹⁷O-decoupled proton MR spectroscopy and imaging with a double-tuned radiofrequency (RF) coil at 2 T was used to detect and quantify H₂ ¹⁷O in tissue phantoms containing various concentrations of ¹⁷O-enriched water in 5% gelatin. The pulse sequence used in these experiments consisted of a conventional proton spinecho sequence with RF irradiation at the ¹⁷O resonance frequency applied between the proton 90° pulse and the signal acquisition window. The double-tuned coil provided several advantages over systems using separate RF coils for ¹⁷O decoupling and proton excitation/detection, including ensuring that the same (or similar) sample volumes are excited and decoupled and permitting accurate calibration of the ¹⁷O decoupling pulse amplitude. The efficiency of ¹⁷O decoupling as a function of decoupling RF amplitude, decoupling duration, and decoupling resonance offset was investigated. Finally, the specific absorption rate of the ¹⁷O decoupled pulse sequence was investigated and found to lie within federal guidelines at 1.5 T.     

Nuclear magnetic relaxation,correlation time spectrum,and molecular dynamics in a linear polymer

The pulsed nuclear magnetic resonance (NMR) method at a proton frequency of 25 MHz at temperatures of 22–160°C is used to detect the transverse magnetization decay in polyisoprene rubbers with various molecular masses, to determine the NMR damping time T ₂, and to measure spin-lattice relaxation time T ₁ and time T _2eff of damping of solid-echo signals under the action of a sequence of MW-4 pulses modified by introducing 180° pulses. The dispersion dependences of T _2eff obtained for each temperature are combined into one using the temperature-frequency equivalence principle. On the basis of the combined dispersion dependence of T _2eff and the data on T ₂ and T ₁, the correlation time spectrum of molecular movements is constructed. Analysis of the shape of this spectrum shows that the dynamics of polymer molecules can be described in the first approximation by the Doi-Edwards tube-reptation model.     

A "magic sandwich" pulse sequence with reduced offset dependence for high-resolution separated local field spectroscopy

A pulse sequence for high resolution separated local field spectroscopy based on "magic sandwich" elements is demonstrated on a single crystal sample. Simulations and experimental results show that this pulse sequence has a reduced frequency offset dependence compared to PISEMA (polarization inversion spin exchange at the magic angle). As a result, it has a larger effective range of homonuclear decoupling, reduced zero-frequency spectral distortions, and more reliable scale factors for individual resonances. In addition, it is easier to setup on commercial spectrometers.     

Improved proton spectroscopic U-FLARE imaging for the detection of coupled resonances in the rat brain in vivo

Modifications of the pulse sequence for spectroscopic U-FLARE imaging are discussed to detect not only the predominant singlet signals of N-acetylaspartate, total creatine, and choline containing compounds or the doublet signal of lactate, but also the coupled resonances of glutamate, glutamine, taurine and myo-inositol. Effective homonuclear decoupling is achieved by use of constant time chemical shift encoding. A maximum signal-to-noise ratio (SNR) can be obtained for a certain coupled resonance of interest by optimizing the evolution period t(c) of the J modulated spin echo. Good reproducibility and a high SNR were achieved by combining several methods for water suppression and by using the displaced variant of U-FLARE. Measurements of a 3 mm slice of the rat brain were performed in vivo within 4 min, giving a nominal voxel size of 1.5 x 1.5 x 3.0 mm3 or 1.5 x 0.75 x 3.0 mm3. Thus, optimized spectroscopic U-FLARE is a powerful tool for proton spectroscopic imaging with high spectral, spatial and temporal resolution.     

Three-dimensional 13C shift/1H-15N coupling/15N shift solid-state NMR correlation spectroscopy.

Triple-resonance experiments capable of correlating directly bonded and proximate carbon and nitrogen backbone sites of uniformly 13C- and 15N-labeled peptides in stationary oriented samples are described. The pulse sequences integrate cross-polarization from 1H to 13C and from 13C to 15N with flip-flop (phase and frequency switched) Lee-Goldburg irradiation for both 13C homonuclear decoupling and 1H-15N spin exchange at the magic angle. Because heteronuclear decoupling is applied throughout, the three-dimensional pulse sequence yields 13C shift/1H-15N coupling/15N shift correlation spectra with single-line resonances in all three frequency dimensions. Not only do the three-dimensional spectra correlate 13C and 15N resonances, they are well resolved due to the three independent frequency dimensions, and they can provide up to four orientationally dependent frequencies as input for structure determination. These experiments have the potential to make sequential backbone resonance assignments in uniformly 13C- and 15N-labeled proteins.     

Three-Dimensional C Shift/H–N Coupling/N Shift Solid-State NMR Correlation Spectroscopy

Triple-resonance experiments capable of correlating directly bonded and proximate carbon and nitrogen backbone sites of uniformly ¹³C- and ¹⁵N-labeled peptides in stationary oriented samples are described. The pulse sequences integrate cross-polarization from ¹H to ¹³C and from ¹³C to ¹⁵N with flip-flop (phase and frequency switched) Lee–Goldburg irradiation for both ¹³C homonuclear decoupling and ¹H–¹⁵N spin exchange at the magic angle. Because heteronuclear decoupling is applied throughout, the three-dimensional pulse sequence yields ¹³C shift/¹H–¹⁵N coupling/¹⁵N shift correlation spectra with single-line resonances in all three frequency dimensions. Not only do the three-dimensional spectra correlate ¹³C and ¹⁵N resonances, they are well resolved due to the three independent frequency dimensions, and they can provide up to four orientationally dependent frequencies as input for structure determination. These experiments have the potential to make sequential backbone resonance assignments in uniformly ¹³C- and ¹⁵N-labeled proteins.     

High resolution 2D 1H-13C correlation of cholesterol in model membrane

High resolution 2D NMR MAS spectra of liposomes, in particular 1H-13C chemical shifts correlations have been obtained on fluid lipid bilayers made of pure phospholipids for several years. We have investigated herein the possibility to obtain high resolution 2D MAS spectra of cholesterol embedded in membranes, i.e. on a rigid molecule whose dynamics is characterized mainly by axial diffusion without internal segmental mobility. The efficiency of various pulse sequences for heteronuclear HETCOR has been compared in terms of resolution, sensitivity and selectivity, using either cross polarization or INEPT for coherence transfer, and with or without MREV-8 homonuclear decoupling during t1. At moderately high spinning speed (9 kHz), a similar resolution is obtained in all cases (0.2 ppm for 1H(3,4), 0.15 ppm for 13C(3,4) cholesterol resonances), while sensitivity increases in the order: INEPT < CP(x4) < CP + MREV. At reduced spinning speed (5 kHz), the homonuclear dipolar coupling between the two geminal protons attached to C(4) gives rise to spinning sidebands from which one can estimate a H-H dipolar coupling of 10 kHz which is in good agreement with the known dynamics of cholesterol in membranes.     

MRICOM–MRI COntrast Modelling using 2D T1–T2 correlation spectra and relaxation signatures

Image contrast is calculated by inputting experimental 2D T₁–T₂ relaxation spectra into the ODIN software interface. The method involves characterising a magnetic resonance imaging pulse sequence with a “relaxation signature” which describes the sensitivity of the sequence to relaxation and is independent of sample parameters. Maximising (or minimising) the overlap between the experimental 2D T₁–T₂ relaxation spectra and the relaxation signature can then be used to maximise image contrast. The concept is illustrated using relaxation signatures for the echo planar imaging and Turbo spin-echo imaging sequences, together with in-vitro 2D T₁–T₂ spectra for liver and cartilage.     

In vivo lactate editing in single voxel proton spectroscopy and proton spectroscopic imaging by homonuclear polarisation transfer

Volume-selective lactate editing has been performed successfully in vitro and in vivo in the brain on a clinical scanner using a PRESS-based single voxel ¹H spectroscopy and a ¹H spectroscopic imaging sequence. The PRESS sequence was made sensitive to homonuclear polarisation by replacing the standard 180° refocusing pulses with 90° pulses. Two acquisitions were made at a total echo time around 2/J (J is the coupling constant for CH and CH₃ spins in lactate ≈7 Hz) whose individual echo times differed by 5.5 ms. Subtraction of one signal from the other yielded the lactate resonance alone. The technique is an effective method of separating the overlapping signals of lactate and lipids. Furthermore this editing method can be performed without state of the art MRI scanner hardware.     

H Chemical Shielding Anisotropies from Polycrystalline Powders Using MSHOT-3 Based CRAMPS

It is demonstrated that combined rotation and multiple-pulse spectroscopy (CRAMPS) based on MSHOT-3 homonuclear multiple-pulse decoupling represents a powerful method for determination of¹H chemical shielding anisotropies from polycrystalline powders. By virtue of high-order dipolar decoupling, large spectral width, resonance offset stability, and the absence of artifacts fromtilted-axis precession, MSHOT-3-based CRAMPS enables straightforward sampling of high-quality spectra. Comparison with explicit calculations, taking the effect of the multiple-pulse sequence into account, shows that the spectra may be simulated and iteratively fitted using standard software for the calculation of magic-angle spinning spectra influenced by chemical shielding anisotropy with the shielding interaction reduced by the scaling factor of the MSHOT-3 decoupling sequence. The method is demonstrated by experimental determination of¹H chemical shielding anisotropies for adipic acid, Ca(OH)₂, malonic acid, and KHSO₄. The data are compared with those determined previously from single-crystal NMR studies.     

Radiofrequency pulse sequences which compensate their own imperfections. 1980

Radiofrequency pulse sequences are described which have the same overall effect as a single 90° or 180° pulse but which compensate the undesirable effects of resonance offset and spatial inhomogeneity of the radiofrequency field H₁. These “composite” pulses are built up from a small number of conventional pulses which rotate the nuclear magnetization vectors about different axes in the rotating frame, while in the intervals between pulses a limited amount of free precession may be allowed to occur. Insight into the way in which pulse imperfections are compensated is obtained by computer simulation of trajectories of families of nuclear spin “isochromats” representing a distribution of H₁ intensity or resonance offset. Composite 90° pulses are suggested as a method of reducing systematic errors in spin-lattice relaxation times derived from progressive saturation or saturation-recovery experiments, and as the preparation pulse of a spin-locking experiment. A test of the effectiveness of the composite 180° pulse sequence has been made by using it for population inversion in a spin-lattice relaxation measurement, where T₁ is derived from the null point in the recovery curve, a technique known to be very sensitive to pulse imperfections.     

Improvement of homonuclear dipolar decoupling sequences in solid-state nuclear magnetic resonance utilising radiofrequency imperfections

The often annoying imperfections in the phases and pulses of typical radiofrequency multiple-pulse irradiation schemes for homonuclear dipolar decoupling are revisited and analysed here. The analysis is with respect to one such multiple-pulse sequence, namely, the windowed phase-modulated Lee-Goldburg sequence. The error terms in the Hamiltonian due to pulse imperfections may lead to effective rotation of the spins around the z-axis giving rise to image free and high-resolution 1H spectra. Certain precautions to be taken with regard to scale factor estimation are also detailed. The analysis also points out the range of off-set values where the best homonuclear dipolar decoupling performance of a particular pulse scheme may be obtained.     

Calculation of Double-Quantum-Coherence Two-dimensional Spectra: Distance Measurements and Orientational Correlations

The double-quantum-coherence (DQC) echo signal for two coupled nitroxides separated by distances ?10 Å, is calculated rigorously for the six-pulse sequence. Successive application of six pulses on the initial density matrix, with appropriate inter-pulse time evolution and coherence pathway selection leaves only the coherent pathways of interest. The amplitude of the echo signal following the last π pulse can be used to obtain a one-dimensional (1D) dipolar spectrum (Pake doublet), and the echo envelope can be used to construct the 2D DQC spectrum. The calculations are carried out using the product space spanned by the two electron-spin magnetic quantum numbers m ₁, m ₂ and the two nuclear-spin magnetic quantum numbers M ₁, M ₂, describing, e.g. two coupled nitroxides in bilabeled proteins. The density matrix is subjected to a cascade of unitary transformations taking into account dipolar and electron exchange interactions during each pulse and during the evolution in the absence of a pulse. The unitary transformations use the eigensystem of the effective spin Hamiltonians obtained by numerical matrix diagonalization. Simulations are carried out for a range of dipolar interactions, D, and microwave magnetic field strength B ₁ for both fixed and random orientations of the two ¹⁴N (and ¹⁵N) nitroxides. Relaxation effects were not included. Several examples of 1D and 2D Fourier transforms of the time-domain signals versus dipolar evolution and spin-echo envelope time variables are shown for illustration. Comparisons are made between 1D rigorous simulations and analytical approximations. The rigorous simulations presented here provide insights into DQC electron spin resonance spectroscopy, they serve as a standard to evaluate the results of approximate theories, and they can be employed to plan future DQC experiments.     

Relaxation Effects in a System of a Spin-1/2 Nucleus Coupled to a Quadrupolar Spin Subjected to RF Irradiation: Evaluation of Broadband Decoupling Schemes

We have investigated the suitability and performance of various decoupling methods on systems in which an observed spin-1/2 nucleusI(¹³C or¹⁵N) is scalar-coupled to a quadrupolar spinS(²H). Simulations and experiments have been conducted by varying the strength of the irradiating radiofrequency (RF) field, RF offset, relaxation times, and decoupling schemes applied in the vicinity of theS-spin resonance. TheT₁relaxation of the quadrupolar spin has previously been shown to influence the efficiency of continuous wave (CW) decoupling applied on resonance in such spin systems. Similarly, the performance of broadband decoupling sequences should also be affected by relaxation. However, virtually all of the more commonly used broadband decoupling schemes have been developed without consideration of relaxation effects. As a consequence, it is not obvious how one selects a suitable sequence for decoupling quadrupolar nuclei with exotic relaxation behavior. Herein we demonstrate that, despite its simplicity, WALTZ-16 decoupling is relatively robust under a wide range of conditions. In these systems it performs as well as the more recently developed decoupling schemes for wide bandwidth applications such as GARP-1 and CHIRP-95. It is suggested that in macromolecular motional regimes, broadband deuterium decoupling can be achieved with relatively low RF amplitudes (500–700 Hz) using WALTZ-16 multiple pulse decoupling.     

NMR relaxation study of avocado quality

Four nuclear magnetic resonance relaxation protocols are investigated as potential candidates for off-line and on-line determination of avocado maturity. Two-dimensionalT ₁-T ₂ andT ₂-D correlation spectroscopy provides the most information but is only suitable for off-line quality control. The CPMGT ₂ spectrum gives avocado oil content but requires intensive data processing. Suppression of the tissue water signal byT ₁-Null methods is shown to be unreliable but a new, single-shot pulse sequence which uses diffusive attenuation to suppress the tissue water is shown to give a good correlation with oil content and is suitable for both off-line and on-line implementation.