A low-E magic angle spinning probe for biological solid state NMR at 750 MHz

Crossed-coil NMR probes are a useful tool for reducing sample heating for biological solid state NMR. In a crossed-coil probe, the higher frequency ¹H field, which is the primary source of sample heating in conventional probes, is produced by a separate low-inductance resonator. Because a smaller driving voltage is required, the electric field across the sample and the resultant heating is reduced. In this work we describe the development of a magic angle spinning (MAS) solid state NMR probe utilizing a dual resonator. This dual resonator approach, referred to as “low-E,” was originally developed to reduce heating in samples of mechanically aligned membranes. The study of inherently dilute systems, such as proteins in lipid bilayers, via MAS techniques requires large sample volumes at high field to obtain spectra with adequate signal-to-noise ratio under physiologically relevant conditions. With the low-E approach, we are able to obtain homogeneous and sufficiently strong radiofrequency fields for both ¹H and ¹³C frequencies in a 4 mm probe with a ¹H frequency of 750 MHz. The performance of the probe using windowless dipolar recoupling sequences is demonstrated on model compounds as well as membrane-embedded peptides.     

Temperature accuracy and temperature gradients in solution-state NMR spectrometers

In solution-state NMR spectrometers, there is a systematic deviation between the temperature of the sample and the temperature reported by the spectrometer. In addition, temperature gradients are often present in the sample. The size of both the temperature deviations and the temperature gradients depends on several factors, including the temperature, the flow rate of the heating/cooling gas, and the amount of radiofrequency heating.     

Sensitivity and resolution enhancement in solid-state NMR spectroscopy of bicelles

Magnetically aligned bicelles are becoming attractive model membranes to investigate the structure, dynamics, geometry, and interaction of membrane-associated peptides and proteins using solution- and solid-state NMR experiments. Recent studies have shown that bicelles are more suitable than mechanically aligned bilayers for multidimensional solid-state NMR experiments. In this work, we describe experimental aspects of the natural abundance (13)C and (14)N NMR spectroscopy of DMPC/DHPC bicelles. In particular, approaches to enhance the sensitivity and resolution and to quantify radio-frequency heating effects are presented. Sensitivity of (13)C detection using single pulse excitation, conventional cross-polarization (CP), ramp-CP, and NOE techniques are compared. Our results suggest that the proton decoupling efficiency of the FLOPSY pulse sequence is better than that of continuous wave decoupling, TPPM, SPINAL, and WALTZ sequences. A simple method of monitoring the water proton chemical shift is demonstrated for the measurement of sample temperature and calibration of the radio-frequency-induced heating in the sample. The possibility of using (14)N experiments on bicelles is also discussed.     

Using a cross-coil to reduce RF heating by an order of magnitude in triple-resonance multinuclear MAS at high fields

Four different coil designs for use with MAS in triple-resonance multi-nuclear experiments at high fields are compared, using a combination of finite element analysis (FEA) software and NMR experiments, with respect to RF field strength per unit power and relative sample heating, as governed by mean E/B(1) within the sample region. A commercial FEA package, Microwave Studio 5.1 by Computer Simulation Technology (CST) is shown to obtain remarkably accurate agreement with the experiments in Q(L), L, B, E, and mode frequencies in all cases. A simplified treatment of RF heating in NMR MAS samples is derived and shown to agree with the NMR experimental results within about 10% for two representative stator designs. The coil types studied include: (1) a variable-pitch solenoid outside a ceramic coilform, (2) a conventional solenoid very closely spaced to the MAS rotor, (3) a scroll coil, and (4) a segmented saddle cross coil (XC) for (1)H with an additional solenoid over it for the two lower-frequency channels. The XC/solenoid is shown to offer substantial advantages in reduced decoupler heating, improved S/N, and improved compatibility with multinuclear tuning and high-power decoupling. This seems largely because the division of labor between two orthogonal coils allows them each, and their associated circuitry, to be separately optimized for their respective regimes.     

Resonator with reduced sample heating and increased homogeneity for solid-state NMR

In the application of solid-state NMR to many systems, the presence of radiofrequency (rf) electric fields inside classical solenoidal coils causes heating of lossy samples. In particular, this is critical for proteins in ionic buffers. Rf sample heating increases proportional to frequency which may result in the need to reduce the rf pulse power to prevent partial or total sample deterioration. In the present paper, we propose a multifrequency-tunable NMR resonator where the sample is electrically shielded from the NMR coil by a conductive sheet that increases the magneto-electric ratio. Expressions for the B1 efficiency as function of magnetic and electric filling factors are derived that allow a direct comparison of different resonators. Rf efficiency, homogeneity, signal-to-noise, and rf sample heating are compared. NMR spectra at 700MHz on ethylene glycol, glycine, and a model protein were acquired to compare the resonators under realistic experimental conditions.     

"Cooking the sample": radiofrequency induced heating during solid-state NMR experiments

Dissipation of radiofrequency (RF) energy as heat during continuous wave decoupling in solid-state NMR experiment was examined outside the conventional realm of such phenomena. A significant temperature increase could occur while performing dynamic NMR measurements provided the sample contains polar molecules and the sequence calls for relatively long applications of RF power. It was shown that the methyl flip motion in dimethylsulfone (DMS) is activated by the decoupling RF energy conversion to heat during a CODEX pulse sequence. This introduced a significant bias in the correlation time–temperature dependency measurement used to obtain the activation energy of the motion. By investigating the dependency of the temperature increase in hydrated lead nitrate on experimental parameters during high-power decoupling one-pulse experiments, the mechanisms for the RF energy deposition was identified. The samples were heated due to dissipation of the energy absorbed by dielectric losses, a phenomenon commonly known as “microwave” heating. It was thus established that during solid-state NMR experiments at moderate B₀ fields, RF heating could lead to the heating of samples containing polar molecules such as hydrated polymers and inorganic solids. In particular, this could result in systematic errors for slow dynamics measurements by solid-state NMR.     

An easy way to prepare pressurized glass inserts for MAS rotors

A novel technique to prepare pressurized glass insert samples for MAS rotors is described. In this technique, a small drop of epoxy is added to the tip of a piston and the gas is squeezed into the insert by pressing the piston. The amount of gas, i.e., pressure, in the sample can be controlled by the overall length of an insert test tube. As examples, (129)Xe NMR spectra taken from samples containing xenon gas, xenon gas and liquid crystal, and xenon gas, liquid crystal and porous solid, are shown. In principle, the method is feasible for making any kind of samples into glass inserts.     

Real-time imaging of the spatial distribution of rf-heating in NMR samples during broadband decoupling

Continuous radio-frequency (rf) irradiation during decoupling and spin-lock periods in NMR pulse sequences may lead to undesired sample heating. Heat-sensitive samples can suffer damage from the sudden temperature rise which cannot be adequately compensated by the temperature control system. Moreover, as the heating is spatially inhomogeneous, higher temperature increases can arise locally than are indicated by the average increase detected by the temperature controller. In this work we present a technique that allows measurement of a real-time 2D-image of the temperature distribution inside an NMR sample during an experiment involving rf-heating. NMR imaging methods have previously been used to project the temperature distribution inside an NMR sample onto a single spatial axis or to acquire steady-state 2D- temperature distributions. The real-time 2D-temperature profiles obtained with our procedure provide much more detailed data. Our results show, that not only inhomogeneous heating but also inhomogeneous sample cooling contribute to the build-up of temperature gradients across the sample. The technique can be used to visualize rf-heating in order to protect sensitive samples and to experimentally test new coil geometries or to guide probehead design.     

Enhanced EPR sensitivity from a ferroelectric cavity insert.

We report the development of a simple ferroelectric cavity insert that increases the electron paramagnetic resonance (EPR) sensitivity by an order of magnitude when a sample is placed within it. The insert is a hollow cylinder (length 4.8 mm, outside diameter 1.7 mm, inside diameter 0.6 mm) made from a single crystal of KTaO(3), which has a dielectric constant of 230 at X-band (9.5 GHz). Its outside dimensions were chosen to produce a resonant frequency in the X-band range, based on electromagnetic field modeling calculations. The insert increases the microwave magnetic field (H(1)) at the center of the insert by a factor of 7.4 when placed in an X-band TM(110) cavity. This increases the EPR signal for a small (volume 0.13 microL) unsaturated nitroxide spin label sample by a factor of 64 at constant microwave power, and by a factor of 9.8 at constant H(1). The insert does not significantly affect the cavity quality factor Q, indicating that this device simply redistributes the microwave fields within the cavity, focusing H(1) onto the sample inside the insert, thus increasing the filling factor. A similar signal enhancement is obtained in the TM(110) and TE(102) cavities, and when the insert is oriented either vertically (parallel to the microwave field) or horizontally (parallel to the DC magnetic field) in the TM(110) cavity. This order-of-magnitude sensitivity enhancement allows EPR spectroscopy to be performed in conventional high-Q cavities on small EPR samples previously only measurable in loop-gap or dielectric resonators. This is of particular importance for small samples of spin-labeled biomolecules.     

Analysis of RF heating and sample stability in aligned static solid-state NMR spectroscopy

Sample instability during solid-state NMR experiments frequently arises due to RF heating in aligned samples of hydrated lipid bilayers. A new, simple approach for estimating sample temperature is used to show that, at 9.4 T, sample heating depends mostly on (1)H decoupling power rather than on (15)N irradiation in PISEMA experiments. Such heating for different sample preparations, including lipid composition, salt concentration and hydration level was assessed and the hydration level was found to be the primary parameter correlated with sample heating. The contribution to RF heating from the dielectric loss appears to be dominant under our experimental conditions. The heat generated by a single scan was approximately calculated from the Q values of the probe, to be a 1.7 degrees C elevation per single pulse sequence iteration under typical sample conditions. The steady-state sample temperature during PISEMA experiments can be estimated based on the method presented here, which correlates the loss factor with the temperature rise induced by the RF heating of the sample.     

生物固体核磁共振中样品发热的研究进展

近年来,固体核磁共振被广泛应用于膜蛋白、纤维化蛋白等体系的结构和功能研究．在固体核磁共振实验中,快速魔角旋转或高功率射频场照射等实验条件将导致样品发热．生物样品发热能导致严重的后果,例如样品温度的快速升高,信号分辨率、信噪比的降低,发热严重时甚至导致样品的不可逆损坏．近年来,人们对样品发热问题进行了一些研究,发现通过优化样品制备条件或固体核磁共振实验条件,以及改进探头设计等手段,可以在一定程度上减轻样品发热．该文主要综述了生物固体核磁共振研究中导致样品发热的原因和减轻样品发热的方法．     

Reducing the NMR sample volume using a single organic liquid: Increased sensitivity for mass-limited samples with standard NMR probes

A simple inexpensive protocol for confining an aqueous sample to the active region of a standard NMR probe is examined for high-resolution NMR. The aqueous sample is sandwiched between an inert perfluorinated organic liquid that has been exploited in the design of micro-coil NMR probes. The procedure is demonstrated with 3 mm NMR tubes at ambient and elevated temperatures but should be equally applicable to smaller diameter tubes. It is shown that confinement has minimal effects on line shape and provides at least a two fold increase in sensitivity over a conventional sample, for the same mass of solute.     

Construction and performance of an NMR tube with a sample cavity formed within magnetic susceptibility-matched glass

We describe the construction and performance of an NMR tube with a magnetic susceptibility matched sample cavity that confines the solution within the detection zone in the axial direction and in a quasi-rectangular region in the radial direction. The slot-like sample cavity provides both good sample volume efficiency and tolerance to sensitivity loss in the sample space. The signal-to-noise ratio per unit volume of the constructed tube was 2.2 times higher than that of a cylindrical tube of 5mm outer diameter with a sample containing 300 mM NaCl at a static magnetic field of 14.1T. Even the overall signal-to-noise ratio of the slot tube was 35% higher than that of the conventional 5mm tube for a sample containing 300 mM NaCl. Similar improvements over existing sample tube geometries were obtained at 950 MHz. Moreover the temperature rise resulting from RF heating was found to be significantly lower for the slot tube even when compared to 3 and 4mm outer diameter cylindrical tubes as measured in a 5mm cryoprobe. A further advantage of this type of tube is that a sample cavity of any desired size and shape can be formed within a cylindrical tube for use in a single cryogenic probe.     

A Modified Alderman–Grant Coil makes possible an efficient cross-coil probe for high field solid-state NMR of lossy biological samples

The design, construction, and performance of a cross-coil double-resonance probe for solid-state NMR experiments on lossy biological samples at high magnetic fields are described. The outer coil is a Modified Alderman–Grant Coil (MAGC) tuned to the ¹H frequency. The inner coil consists of a multi-turn solenoid coil that produces a B₁ field orthogonal to that of the outer coil. This results in a compact nested cross-coil pair with the inner solenoid coil tuned to the low frequency detection channel. This design has several advantages over multiple-tuned solenoid coil probes, since RF heating from the ¹H channel is substantially reduced, it can be tuned for samples with a wide range of dielectric constants, and the simplified circuit design and high inductance inner coil provides excellent sensitivity. The utility of this probe is demonstrated on two electrically lossy samples of membrane proteins in phospholipid bilayers (bicelles) that are particularly difficult for conventional NMR probes. The 72-residue polypeptide embedding the transmembrane helices 3 and 4 of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) (residues 194–241) requires a high salt concentration in order to be successfully reconstituted in phospholipid bicelles. A second application is to paramagnetic relaxation enhancement applied to the membrane-bound form of Pf1 coat protein in phospholipid bicelles where the resistance to sample heating enables high duty cycle solid-state NMR experiments to be performed.     

Using heat to control the sample spinning speed in MAS NMR

A new approach using temperature to control the spinning speed of a sample rotor in magic-angle spinning NMR is presented. Instead of an electro-mechanical valve that regulates the flow of drive gas to control the spinning speed in traditional MAS NMR systems, we use a small heater wire located directly in the stator. The sample spinning speed is controlled very accurately with a surprisingly low heating power of 1 W. Results on a benchtop unit demonstrate the capability of the system.     

Heating of samples induced by fast magic-angle spinning

Intense sample heating through high-speed magic-angle spinning (MAS; up to 58 K temperature difference) is demonstrated. The role of probehead and spinner design, as well as that of the temperature of the bearing air on the heating of a rotating sample, is examined. MAS-induced heating can affect the accurate determination of the isotropic value of the chemical shift as well as the principal values, asymmetry and anisotropy parameters of the chemical shift tensor. In some cases, a very large temperature gradient (12 K) within the fast rotating sample was found, which may limit the resolution of high-speed 1H MAS nuclear magnetic resonance (NMR) spectra.     

T1-relaxation of 129Xe on metal single crystal surfaces-multilayer experiments on iridium and monolayer considerations

The surface of a typical laboratory single crystal has about 10(15) surface atoms or adsorption sites, respectively, and is thus far out of reach for conventional NMR experiments using thermal polarization. It should however be in reach for NMR of adsorbed laser polarized (hyperpolarized) 129Xe, which is produced by spin transfer from optically pumped rubidium. With multilayer experiments of xenon adsorbed on an iridium surface we do not only demonstrate that monolayer sensitivity has been obtained, we also show that such surface experiments can be performed under ultra high vacuum conditions with the crystal being mounted in a typical surface analysis chamber on a manipulator with far-reaching sample heating and cooling abilities. With only four spectra summed up we present an NMR signal from at most 4x10(14) atoms of 129Xe, four layers of naturally abundant xenon, respectively. The fact that no monolayer signal has been measured so far is explained by a fast Korringa relaxation due to the Fermi contact interaction of the 129Xe nuclei with the electrons of the metal substrate. T(1)-relaxation times in the order of several ms have been calculated using all electron density functional theory for several metal substrates.     

Measurement of sample temperatures under magic-angle spinning from the chemical shift and spin-lattice relaxation rate of 79Br in KBr powder

Accurate determination of sample temperatures in solid state nuclear magnetic resonance (NMR) with magic-angle spinning (MAS) can be problematic, particularly because frictional heating and heating by radio-frequency irradiation can make the internal sample temperature significantly different from the temperature outside the MAS rotor. This paper demonstrates the use of (79)Br chemical shifts and spin-lattice relaxation rates in KBr powder as temperature-dependent parameters for the determination of internal sample temperatures. Advantages of this method include high signal-to-noise, proximity of the (79)Br NMR frequency to that of (13)C, applicability from 20 K to 320 K or higher, and simultaneity with adjustment of the MAS axis direction. We show that spin-lattice relaxation in KBr is driven by a quadrupolar mechanism. We demonstrate a simple approach to including KBr powder in hydrated samples, such as biological membrane samples, hydrated amyloid fibrils, and hydrated microcrystalline proteins, that allows direct assessment of the effects of frictional and radio-frequency heating under experimentally relevant conditions.     

Using low-E resonators to reduce RF heating in biological samples for static solid-state NMR up to 900 MHz

RF heating of solid-state biological samples is known to be a destabilizing factor in high-field NMR experiments that shortens the sample lifetime by continuous dehydration during the high-power cross-polarization and decoupling pulses. In this work, we describe specially designed, large volume, low-E 15N-1H solid-state NMR probes developed for 600 and 900 MHz PISEMA studies of dilute membrane proteins oriented in hydrated and dielectrically lossy lipid bilayers. The probes use an orthogonal coil design in which separate resonators pursue their own aims at the respective frequencies, resulting in a simplified and more efficient matching network. Sample heating at the 1H frequency is minimized by a loop-gap resonator which produces a homogeneous magnetic field B1 with low electric field E. Within the loop-gap resonator, a multi-turn solenoid closely matching the shape of the sample serves as an efficient observe coil. We compare power dissipation in a typical lossy bilayer sample in the new low-E probe and in a previously reported 15N-1H probe which uses a double-tuned 4-turn solenoid. RF loss in the sample is measured in each probe by observing changes in the 1H 360 degrees pulse lengths. For the same values of 1H B1 field, sample heating in the new probe was found to be smaller by an order of magnitude. Applications of the low-E design to the PISEMA study of membrane proteins in their native hydrated bilayer environment are demonstrated at 600 and 900 MHz.     

SQUID-detected microtesla MRI in the presence of metal

In magnetic resonance imaging performed at fields of 1 T and above, the presence of a metal insert can distort the image because of susceptibility differences within the sample and modification of the radiofrequency fields by screening currents. Furthermore, it is not feasible to perform nuclear magnetic resonance (NMR) spectroscopy or acquire a magnetic resonance image if the sample is enclosed in a metal container. Both problems can be overcome by substantially lowering the NMR frequency. Using a microtesla imaging system operating at 2.8 kHz, with a superconducting quantum interference device as the signal detector, we have obtained distortion-free images of a phantom containing a titanium bar and three-dimensional images of an object enclosed in an aluminum can; in both cases high-field images are inaccessible.