Improved Spin-Echo-Edited NMR Diffusion Measurements

The need for simple and robust schemes for the analysis of ligand-protein binding has resulted in the development of diffusion-based NMR techniques that can be used to assay binding in protein solutions containing a mixture of several ligands. As a means of gaining spectral selectivity in NMR diffusion measurements, a simple experiment, the gradient modified spin-echo (GOSE), has been developed to reject the resonances of coupled spins and detect only the singlets in the (1)H NMR spectrum. This is accomplished by first using a spin echo to null the resonances of the coupled spins. Following the spin echo, the singlet magnetization is flipped out of the transverse plane and a dephasing gradient is applied to reduce the spectral artifacts resulting from incomplete cancellation of the J-coupled resonances. The resulting modular sequence is combined here with the BPPSTE pulse sequence; however, it could be easily incorporated into any pulse sequence where additional spectral selectivity is desired. Results obtained with the GOSE-BPPSTE pulse sequence are compared with those obtained with the BPPSTE and CPMG-BPPSTE experiments for a mixture containing the ligands resorcinol and tryptophan in a solution of human serum albumin.     

The matrix formalism for generalised gradients with time-varying orientation in diffusion NMR

Protein backbone 15N NMR spin relaxation rates are useful in characterizing the protein dynamics and structures. To observe the protein nuclear-spin resonances a pulse sequence has to include a water suppression scheme. There are two commonly employed methods, saturating or dephasing the water spins with pulse field gradients and keeping them unperturbed with flip-back pulses. Here different water suppression methods were incorporated into pulse sequences to measure 15N longitudinal T1 and transversal rotating-frame T1ρ spin relaxation. Unexpectedly the 15N T1 relaxation time constants varied significantly with the choice of water suppression method. For a 25-kDa Escherichiacoli. glutamine binding protein (GlnBP) the T1 values acquired with the pulse sequence containing a water dephasing gradient are on average 20% longer than the ones obtained using a pulse sequence containing the water flip-back pulse. In contrast the two T1ρ data sets are correlated without an apparent offset. The average T1 difference was reduced to 12% when the experimental recycle delay was doubled, while the average T1 values from the flip-back measurements were nearly unchanged. Analysis of spectral signal to noise ratios (s/n) showed the apparent slower 15N relaxation obtained with the water dephasing experiment originated from the differences in 1HN recovery for each relaxation time point. This in turn offset signal reduction from 15N relaxation decay. The artifact becomes noticeable when the measured 15N relaxation time constant is comparable to recycle delay, e.g., the 15N T1 of medium to large proteins. The 15N relaxation rates measured with either water suppression schemes yield reasonable fits to the structure. However, data from the saturated scheme results in significantly lower Model-Free order parameters (=0.81) than the non-saturated ones (=0.88), indicating such order parameters may be previously underestimated.     

基于选择编码的超快速磁共振波谱方法

核磁共振(NMR)波谱技术是当今最有力的谱学工具之一,在化学、生物和医药等众多领域获得重要而广泛的应用．基于时空编码的快速采样方法自2002年Frydman小组提出后,大大增强了高维磁共振波谱的采样效率．在某一些应用体系中,存在若干个强度远超于其他谱峰的情况,很容易由于动态增益不足而检测不到某些较弱的谱峰,而往往这些较弱的谱峰包含着感兴趣的信息．且在实际的化学生物应用中,存在选择性感兴趣检测的情况,即只需要选择性地观察若干个具有标记作用的谱峰．由于时空编码技术借助于高速切换的双极性梯度来完成解码,因而无法选择性地检测若干个非连续的频点．为解决以上两个问题,该文提出一种选择编码的时空编码方法,即在序列中施加选择性脉冲,选择性破坏某些谱峰的编码过程,使之不能在解码期解码,从而简化谱图,实现选择性压制或者非连续频点的感兴趣检测．如果把选择性反转脉冲换为硬反转脉冲加选择性反转脉冲,则最终的谱图中只出现被选择性脉冲选中的谱峰．理论分析及相关的实验验证了这种方法的可行性和有效性.     

A method for detection of spectral spin diffusion from minor peaks, and its application to F MAS NMR of antimony(III)-doped fluorapatite

A new technique for detecting spectral spin diffusion in solids under MAS NMR conditions that is particularly well suited for accurately measuring cross-relaxation from minor spectral components is presented. The pulse sequence, SINK (Saturation Inter-Nuclear Kinetics), selectively saturates the magnetization of a minor spectral component with a series of rotor-synchronized DANTE pulse trains and monitors spin diffusion to other peaks with a non-selective 90° pulse. We have used SINK with ¹⁹F MAS NMR on samples of calcium fluorapatite doped with Sb³⁺ to measure spin diffusion between a weak peak at 68.6 ppm due to fluoride ions associated with Sb³⁺ and other peaks in the spectrum. The SINK experiment clearly demonstrates that spin diffusion from the former peak to the main resonance of fluorapatite at 64.0 ppm is faster than spin diffusion to a second antimony-related peak at 65.6 ppm. These results strengthen our previous conclusion that antimony(III) occupies a phosphate site in the apatite lattice, with an SbO₃³⁻ group replacing a PO₄³⁻ group. The SINK experiment also enables the detection of a “hidden” peak at approximately 62.9 ppm that is otherwise obscured by the intense main peak at 64.0 ppm.     

Ultrabroadband background-free coherent anti-Stokes Raman scattering microscopy based on a compact Er:fiber laser system

We demonstrate a scheme for efficient coherent anti-Stokes Raman scattering (CARS) microscopy free of nonresonant background. Our method is based on a compact Er:fiber laser source. Impulsive excitation of molecular resonances is achieved by an 11 fs pulse at 1210 nm. Broadband excitation gives access to molecular resonances from 0 cm(-1) up to 4000 cm(-1). Time-delayed narrowband probing at 775 nm enables sensitive and high-speed spectral detection of the CARS signal free of nonresonant background with a resolution of 10 cm(-1).     

Diffusion and relaxation effects in general stray field NMR experiments

We analyze the evolution of magnetization following any series of radiofrequency pulses in strongly inhomogeneous fields, with particular attention to diffusion and relaxation effects. When the inhomogeneity of the static magnetic field approaches or exceeds the strength of the RF field, the magnetization has contributions from different coherence pathways. The diffusion or relaxation induced decay of the signal amplitude is in general nonexponential, even if the sample has single relaxation times T(1), T(2) and a single diffusion coefficient D. In addition, the shape of the echo depends on diffusion and relaxation. It is possible to separate contributions from different coherence pathways by phase cycling of the RF pulses. The general analysis is tested on stray field measurements using two different pulse sequences. We find excellent agreement between measurements and calculations. The inversion recovery sequence is used to study the relaxation effects. We demonstrate two different approaches of data analysis to extract the relaxation time T(1). Finite pulse width effects on the timing of the echo formation are also studied. Diffusion effects are analyzed using the Carr--Purcell--Meiboom--Gill sequence. In a stray field of a constant gradient g, we find that unrestricted diffusion leads to nonexponential signal decay versus echo number N, but within experimental error the diffusion attenuation is still only a function of g(2)Dt(3)(E)N, where t(E) is the echo spacing.     

Application of multiple inversion recovery for suppression of macromolecule resonances in short echo time (1)H NMR spectroscopy of human brain.

Macromolecules contribute broad "background" resonances to the (1)H NMR brain spectra at short echo times. The application of long echo times is the most widely used method for removing these resonances. Here, it is demonstrated that these background resonances may be suppressed at short echo times using multiple inversion recovery (MIR). In the technique presented, the MIR sequence consists of four adiabatic inversion pulses, applied preparatory to a 20-ms echo time stimulated echo localization sequence. The inversion times (359, 157, 69, and 20 ms) were selected to preferentially suppress macromolecules with longitudinal relaxation times between 38 and 300 ms. While the resulting spectra have lower overall signal-to-noise, baseline contributions from macromolecules are greatly reduced. Unlike the typical long TE acquisitions, the short TE MIR acquisition preserves the myo-inositol resonance.     

改进的ROESY技术及实验方法

本文介绍了对ROESY(CAMELSPIN)技术改进的脉冲序列的原理。这种改进的技术能有效的抑制偏共振效应的影响和提高谱的灵敏度。进行了1D和2D ROESY实验。实验结果表明,改进的ROESY实验能提高谱图灵敏度40%～80%,并能有效地压制其它2D响应信号。     

Analysis of protein/ligand interactions with NMR diffusion measurements: the importance of eliminating the protein background

Pulsed-field gradient nuclear magnetic resonance (PFG-NMR) is a well-established method for the determination of translational diffusion coefficients. Recently, this method has found applicability in the combinatorial arena with the introduction of affinity NMR for characterizing protein/ligand interactions. Although affinity NMR has been reported to be an effective method for the identification of active compounds in a complex mixture, there are limitations of this method. We have developed a simple mathematical model to predict optimum concentration ratios of the ligand and protein in order to observe maximum changes in the ligand diffusion coefficient upon protein binding. The ligand/protein systems of L-tryptophan and ibuprofen binding to human serum albumin were chosen to demonstrate the usefulness of this model. However, even when the conditions of the mathematical model are satisfied, the spectral background arising from the protein in proton-detected experiments can be problematic. To this end, we have employed spectral subtraction of the protein spectrum to yield ligand diffusion coefficients that are in agreement with those predicted by simulation.     

Self-diffusion measurements by a mobile single-sided NMR sensor with improved magnetic field gradient

A simple and fast method of measuring self-diffusion coefficients of protonated systems with a mobile single-sided NMR sensor is discussed. The NMR sensor uses a magnet geometry that generates a highly flat sensitive volume where a strong and highly uniform static magnetic field gradient is defined. Self-diffusion coefficients were measured by Hahn- and stimulated echoes detected in the presence of the uniform magnetic field gradient of the static field. To improve the sensitivity of these experiments, a Carr-Purcell-Meiboom-Gill pulse sequence was applied after the main diffusion-encoding period. By adding the echo train the experimental time was strongly shortened, allowing the measurement of complete diffusion curves in less than 1min. This method has been tested by measuring the self-diffusion coefficients D of various organic solvents and poly(dimethylsiloxane) samples with different molar masses. Diffusion coefficients were also measured for n-hexane absorbed at saturation in natural rubber with different cross-link densities. The results show a dependence on the concentration that is in good agreement with the theoretical prediction. Moreover, the stimulated-echo sequence was successfully used to measure the diffusion coefficient as a function of the evolution time in systems with restricted diffusion. This type of experiment proves the pore geometry and gives access to the surface-to-volume ratio. It was applied to measure the diffusion of water in sandstones and sheep Achilles tendon. Thanks to the strong static gradient G(0), all diffusion coefficients could be measured without having to account for relaxation during the pulse sequence.     

Diffusion Measurements Using Radiofrequency Field Gradient: Artifacts, Remedies, Practical Hints

The two major advantages of experiments carried out with radiofrequency (RF) field-gradient NMR are the instrumental simplicity and the insensitivity to background static magnetic field gradients. These features combined with large RF gradients, which became available only recently, should make this technique especially attractive for molecular translational diffusion studies. However, a critical evaluation of the method shows that under some circumstances (small and/or heterogeneous samples, weak diffusion coefficients, very short relaxation times) the quality of measurements may be affected by a number of artifacts. Their origin has been investigated and several remedies have been considered; in particular, a new improved sequence is presented. The success of various experimental tests demonstrates the efficiency of the proposed solutions which thus open the way to much wider application fields.     

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.     

Accurate intensities of broad NMR lines from composite pulse experiments

Accurate determination of integral intensities of broad lines is difficult when spin relaxation during the applied pulses cannot be neglected and/or when ringing of the tank circuit interferes with the signal. Here we present an extension of the analytical solution of the generalized Bloch equations (G. A. Morris and P. B. Chilvers, J. Magn. Reson. A 107, 236 (1994)), which is then used to evaluate the signal intensity obtained in a composite pulse experiment designed to cancel ringing effects. Comparing intensities of broad and narrow (81)Br spectral lines tests and proves the accuracy of this approach.     

Determination of water self-diffusion coefficient in complex food products by low field 1H PFG-NMR: comparison between the standard spin-echo sequence and the T1-weighted spin-echo sequence

In 1990, Van Den Enden et al. proposed a method for the determination of water droplet size distributions in emulsions using a pulsed-field-gradient nuclear magnetic resonance (PFG-NMR) T1-weighted stimulated-echo technique. This paper describes both the T1-weighted spin-echo sequence, an improved method based on this earlier work, and, the standard PFG spin-echo sequence. These two methods were compared for water self-diffusion coefficient measurement in the fatty protein concentrate sample used as a 'cheese model.' The transversal and longitudinal relaxation parameters T1 and T2 were determined according to the temperature and investigated for each sample; fat-free protein concentrate sample, pure anhydrous milk fat, and fatty protein concentrate sample. The water self-diffusion in fat-free protein concentrate samples followed a linear behavior. Consequently, the water self-diffusion coefficient could be easily characterized for fat-free protein concentrate samples. However, it seemed more complicated to obtain accurate water self-diffusion in fatty protein concentrate samples since the diffusion-attenuation data were fitted by a bi-exponential function. This paper demonstrates that the implementation of the T1-weighted spin-echo sequence, using the different T1 properties of water and fat phases, allows the accurate determination of water self-diffusion coefficient in a food product. To minimize the contribution of the 1H nuclei in the fat phase on the NMR echo signal, the fat protons were selectively eliminated by an additional 180 degrees pulse. This new method reduces the standard errors of diffusion data obtained with a basic spin-echo technique, by a factor of 10. The effectiveness of the use of the T1-weighted spin-echo sequence to perform accurate water self-diffusion coefficients measurement in fatty products is thus demonstrated.     

A phase cycle scheme that significantly suppresses offset-dependent artifacts in the R2-CPMG 15N relaxation experiment

R2-CPMG 15N relaxation experiments form the basis of NMR dynamics measurements, both for analysis of nano-pico second dynamics and milli-micro second dynamics (kinetics). It has been known for some time that in the practical limit of finite pulse widths, which becomes acute when using cryogenic probes, systematic errors in the apparent R2 relaxation behavior occur for spins far off-resonance from the RF carrier. Inaccurate measurement of R2 rates propagates into quantitative models such as model-free relaxation analysis, rotational diffusion tensor analysis, and relaxation dispersion. The root of the problem stems from evolution of the magnetization vectors out of the XY-plane, both during the pulses as well as between the pulses. These deviations vary as a function of pulse length, number of applied CPMG pulses, and CPMG inter-pulse delay. Herein, we analyze these effects in detail with experimentation, numerical simulations, and analytical equations. Our work suggests a surprisingly simple change in the phase progression of the CPMG pulses, which leads to a remarkable improvement in performance. First, the applicability range of the CPMG experiment is increased by a factor of two in spectral width; second, the dynamical/kinetic processes that can be assessed are significantly extended towards the slower time scale; finally, the robustness of the relaxation dispersion experiments is greatly improved.     

Constant time steady gradient NMR diffusometry using the secondary stimulated echo

A pulse sequence producing a second stimulated echo is suggested for the compensation of relaxation and residual dipolar interaction effects in steady gradient spin echo diffusometry. Steady field gradients of considerable strength exist in the fringe field of NMR magnets, for instance. While the absolute echo time of the second stimulated echo is kept constant throughout the experiment, the interval between the first two radiofrequency pulses is augmented leading to a modulation of the amplitude of that second stimulated echo by self-diffusion only. The unique feature of this technique is that it is of a single-scan/single-echo-signal nature. That is, no reference signals neither of the same pulse sequence nor of separate experiments are needed. The new method was tested with poly(ethylene oxide) melts and proved to provide reliable data for (time dependent) self-diffusion coefficients down to the physical limit (D approximately 10(-15)m(2)/s) when flip-flop spin diffusion starts to become effective.     

Natural abundance (17)O NMR spectroscopy of rat brain in vivo

Oxygen is an abundant element that is present in almost all biologically relevant molecules. NMR observation of oxygen has been relatively limited since the NMR-active isotope, oxygen-17, is only present at a 0.037% natural abundance. Furthermore, as a spin 5/2 nucleus oxygen-17 has a moderately strong quadrupole moment which leads to fairly broad resonances (T(2)=1-4 ms). However, the similarly short T(1) relaxation constants allow substantial signal averaging, whereas the large chemical shift range (>300 ppm) improves the spectral resolution of (17)O NMR. Here it is shown that high-quality, natural abundance (17)O NMR spectra can be obtained from rat brain in vivo at 11.74 T. The chemical shifts and line widths of more than 20 oxygen-containing metabolites are established and the sensitivity and potential for (17)O-enriched NMR studies are estimated.     

The line shapes of the water proton resonances of red blood cells containing carbonyl hemoglobin, deoxyhemoglobin, and methemoglobin: implications for the interpretation of proton MRI at fields of 1.5 T and below

The 300 MHz (7 T) water proton resonances of suspensions of red blood cells containing paramagnetic deoxyhemoglobin or methemoglobin can be resolved into two broad lines assignable to intra- and extracellular water which undergoes rapid T2 relaxation by diffusion in magnetic field gradients induced by the intracellular paramagnets. The width of the resolved lines allowed an estimate of the maximum contribution that diffusion makes to T2 relaxation at 7 T. The dependence of the diffusion contribution on the square of the strength of the static magnetic field suggest that diffusion makes a small contribution to water proton T2 relaxation at 1.5 T compared to 7 T, and a negligible one at 0.5 T in early and intermediate hematomas containing deoxyhemoglobin or methemoglobin in intact red blood cells. At the lower field strengths, water proton T2 relaxation is apparently dominated by the rapid chemical exchange (mean lifetime tau = 10 msec) between the intra- and extracellular environments.