Electron-decoupled MAS DNP with N@C60.

Frequency-chirped microwaves decouple electron- and 13C-spins in magic-angle spinning N@C60:C60 powder, improving DNP-enhanced 13C NMR signal intensity by 12% for 7 s polarization, and 5% for 30 s polarization. This electron decoupling demonstration is a step toward utilizing N@C60 as a controllable electron-spin source for magic-angle spinning magnetic resonance experiments.

In Situ Detection of Endogenous HIV Activation by Dynamic Nuclear Polarization NMR and Flow Cytometry.

We demonstrate for the first time in-cell dynamic nuclear polarization (DNP) in conjunction with flow cytometry sorting to address the cellular heterogeneity of in-cell samples. Utilizing a green fluorescent protein (GFP) reporter of HIV reactivation, we correlate increased 15N resonance intensity with cytokine-driven HIV reactivation in a human cell line model of HIV latency. As few as 10% GFP+ cells could be detected by DNP nuclear magnetic resonance (NMR). The inclusion of flow cytometric sorting of GFP+ cells prior to analysis by DNP-NMR further boosted signal detection through increased cellular homogeneity with respect to GFP expression. As few as 3.6 million 15N-labeled GFP+ cells could be readily detected with DNP-NMR. Importantly, cell sorting allowed for the comparison of cytokine-treated GFP+ and GFP- cells in a batch-consistent way. This provides an avenue for normalizing NMR spectral contributions from background cellular processes following treatment with cellular modulators. We also demonstrate the remarkable stability of AMUPol (a nitroxide biradical) in Jurkat T cells and achieved in-cell enhancements of 46 with 10 mM AMUPol, providing an excellent model system for further in-cell DNP-NMR studies. This represents an important contribution to improving in-cell methods for the study of endogenously expressed proteins by DNP-NMR.

Electron Decoupling with Chirped Microwave Pulses for Rapid Signal Acquisition and Electron Saturation Recovery.

Dynamic nuclear polarization (DNP) increases NMR sensitivity by transferring polarization from electron to nuclear spins. Herein, we demonstrate that electron decoupling with chirped microwave pulses enables improved observation of DNP-enhanced 13 C spins in direct dipolar contact with electron spins, thereby leading to an optimal delay between transients largely governed by relatively fast electron relaxation. We report the first measurement of electron longitudinal relaxation time (T1e ) during magic angle spinning (MAS) NMR by observation of DNP-enhanced NMR signals (T1e =40±6 ms, 40 mM trityl, 4.0 kHz MAS, 4.3 K). With a 5 ms DNP period, electron decoupling results in a 195 % increase in signal intensity. MAS at 4.3 K, DNP, electron decoupling, and short recycle delays improve the sensitivity of 13 C in the vicinity of the polarizing agent. This is the first demonstration of recovery times between MAS-NMR transients being governed by short electron T1 and fast DNP transfer.

Dynamic Nuclear Polarization Nuclear Magnetic Resonance in Human Cells Using Fluorescent Polarizing Agents.

Solid state nuclear magnetic resonance (NMR) enables atomic-resolution characterization of the molecular structure and dynamics within complex heterogeneous samples, but it is typically insensitive. Dynamic nuclear polarization (DNP) increases the NMR signal intensity by orders of magnitude and can be performed in combination with magic angle spinning (MAS) for sensitive, high-resolution spectroscopy. Here we report MAS DNP experiments, for the first time, within intact human cells with >40-fold DNP enhancement and a sample temperature of <6 K. In addition to cryogenic MAS results at <6 K, we also show in-cell DNP enhancements of 57-fold at 90 K. In-cell DNP is demonstrated using biradicals and sterically shielded monoradicals as polarizing agents. A novel trimodal polarizing agent is introduced for DNP, which contains a nitroxide biradical, a targeting peptide for cell penetration, and a fluorophore for subcellular localization with confocal microscopy. The fluorescent polarizing agent provides in-cell DNP enhancements of 63-fold at a concentration of 2.7 mM. These experiments pave the way for structural characterization of biomolecules in an endogenous cellular context.

Magic angle spinning NMR with metallized rotors as cylindrical microwave resonators.

We introduce a novel design for millimeter wave electromagnetic structures within magic angle spinning (MAS) rotors. In this demonstration, a copper coating is vacuum deposited onto the outside surface of a sapphire rotor at a thickness of 50 nm. This thickness is sufficient to reflect 197-GHz microwaves, yet not too thick as to interfere with radiofrequency fields at 300 MHz or prevent sample spinning due to eddy currents. Electromagnetic simulations of an idealized rotor geometry show a microwave quality factor of 148. MAS experiments with sample rotation frequencies of ωr /2π = 5.4 kHz demonstrate that the drag force due to eddy currents within the copper does not prevent sample spinning. Spectra of sodium acetate show resolved 13 C J-couplings of 60 Hz and no appreciable broadening between coated and uncoated sapphire rotors, demonstrating that the copper coating does not prevent shimming and high-resolution nuclear magnetic resonance spectroscopy. Additionally, 13 C Rabi nutation curves of ω1 /2π = 103 kHz for both coated and uncoated rotors indicate no detrimental impact of the copper coating on radio frequency coupling of the nuclear spins to the sample coil. We present this metal coated rotor as a first step towards an MAS resonator. MAS resonators are expected to have a significant impact on developments in electron decoupling, pulsed dynamic nuclear polarization (DNP), room temperature DNP, DNP with low-power microwave sources, and electron paramagnetic resonance detection.

Electron Decoupling with Dynamic Nuclear Polarization in Rotating Solids.

Dynamic nuclear polarization (DNP) can enhance NMR sensitivity by orders of magnitude by transferring spin polarization from electron paramagnetic resonance (EPR) to NMR. However, paramagnetic DNP polarizing agents can have deleterious effects on NMR signals. Electron spin decoupling can mitigate these paramagnetic relaxation effects. We demonstrate electron decoupling experiments in conjunction with DNP and magic-angle-spinning NMR spectroscopy. Following a DNP and spin diffusion period, the microwave irradiation frequency is quickly tuned on-resonance with electrons on the DNP polarizing agent. The electron decoupling performance shows a strong dependence on the microwave frequency and DNP polarization time. Microwave frequency sweeps through the EPR line shape are shown as a time domain strategy to significantly improve electron decoupling. For 13C spins on biomolecules frozen in a glassy matrix, electron decoupling reduces the line widths by 11% (47 Hz) and increases the intensity by 14%.

Describing angular momentum conventions in circularly polarized optically pumped NMR in GaAs and CdTe.

The physical phenomena governing hyperpolarization through optical pumping of conduction electrons continue to be explored in multiple semiconductor systems. One early finding has been the asymmetry between the optically pumped nuclear magnetic resonance (OPNMR) signals when generated by different circular polarizations (i.e., light helicities). Because these resonances are asymmetric, the midpoint between the signals prepared with each of the two circular polarizations is either a positive or negative value, termed an "offset" that is representative of an optical Overhauser enhancement. Both negative offsets (in GaAs) and positive offsets (in CdTe) have been observed. The origins of these offsets in semiconductors are believed to arise from thermalized electrons; however, to the best of our knowledge, no study has systematically tested this hypothesis. To that end, we have adopted two configurations for OPNMR experiments-one in which the Poynting vector of the laser light and magnetic field are parallel, and one in which they are antiparallel, while other experimental conditions are kept the same. We find that the OPNMR signal response to a fixed helicity of light depends on the experimental configuration, and this configuration needs to be accounted for in order to properly describe the OPNMR results. Further, studying the offsets as a function of field strength shows that the optical Overhauser enhancement (the offset) increases in magnitude with field strength. Finally, by describing all angular momentum and phasing conventions unambiguously, we are able to determine that the absorptively-phased appearance of 113Cd (and 125Te) OPNMR in CdTe is a consequence of the sign of the nuclear gyromagnetic ratios for these isotopes.

Characterization of frequency-chirped dynamic nuclear polarization in rotating solids.

Continuous wave (CW) dynamic nuclear polarization (DNP) is used with magic angle spinning (MAS) to enhance the typically poor sensitivity of nuclear magnetic resonance (NMR) by orders of magnitude. In a recent publication we show that further enhancement is obtained by using a frequency-agile gyrotron to chirp incident microwave frequency through the electron resonance frequency during DNP transfer. Here we characterize the effect of chirped MAS DNP by investigating the sweep time, sweep width, center-frequency, and electron Rabi frequency of the chirps. We show the advantages of chirped DNP with a trityl-nitroxide biradical, and a lack of improvement with chirped DNP using AMUPol, a nitroxide biradical. Frequency-chirped DNP on a model system of urea in a cryoprotecting matrix yields an enhancement of 142, 21% greater than that obtained with CW DNP. We then go beyond this model system and apply chirped DNP to intact human cells. In human Jurkat cells, frequency-chirped DNP improves enhancement by 24% over CW DNP. The characterization of the chirped DNP effect reveals instrument limitations on sweep time and sweep width, promising even greater increases in sensitivity with further technology development. These improvements in gyrotron technology, frequency-agile methods, and in-cell applications are expected to play a significant role in the advancement of MAS DNP.

Dynamic Nuclear Polarization with Electron Decoupling in Intact Human Cells and Cell Lysates.

Dynamic nuclear polarization (DNP) is used to improve the inherently poor sensitivity of nuclear magnetic resonance spectroscopy by transferring spin polarization from electrons to nuclei. However, DNP radicals within the sample can have detrimental effects on nuclear spins close to the polarizing agent. Chirped microwave pulses and electron decoupling (eDEC) attenuate these effects in model systems, but this approach is yet to be applied to intact cells or cellular lysates. Herein, we demonstrate for the first time exceptionally fast 1H T1DNP times of just 200 and 300 ms at 90 and 6 K, respectively, using a newly synthesized methylated trityl radical within intact human cells. We further demonstrate that eDEC can also be applied to intact human cells and human and bacterial cell lysates. We investigate eDEC efficiency at different temperatures, with different solvents, and with two trityl radical derivatives. At 90 K, eDEC yields a 13C signal intensity increase of 8% in intact human cells and 10% in human and bacterial cell lysates. At 6 K, eDEC provides larger intensity increases of 15 and 39% in intact human cells and cell lysates, respectively. Combining the manipulation of electron spins with frequency-chirped pulses and sample temperatures approaching absolute zero is a promising avenue for executing rapid, high-sensitivity magic-angle spinning DNP in complex cellular environments.

Sensitivity analysis of magic angle spinning dynamic nuclear polarization below 6 K.

Dynamic nuclear polarization (DNP) improves signal-to-noise in nuclear magnetic resonance (NMR) spectroscopy. Signal-to-noise in NMR can be further improved with cryogenic sample cooling. Whereas MAS DNP is commonly performed between 25 and 110 K, sample temperatures below 6 K lead to further improvements in sensitivity. Here, we demonstrate that solid effect MAS DNP experiments at 6 K, using trityl, yield 3.2× more sensitivity compared to 90 K. Trityl with solid effect DNP at 6 K yields substantially more signal to noise than biradicals and cross effect DNP. We also characterize cross effect DNP with AMUPol and TEMTriPol-1 biradicals for DNP magic angle spinning at temperatures below 6 K and 7 Tesla. DNP enhancements determined from microwave on/off intensities are 253 from AMUPol and 49 from TEMTriPol-1. The higher thermal Boltzmann polarization at 6 K compared to 298 K, combined with these enhancements, should result in 10,000× signal gain for AMUPol and 2000× gain for TEMTriPol-1. However, we show that AMUPol reduces signal in the absence of microwaves by 90% compared to 41% by TEMTriPol-1 at 7 Tesla as the result of depolarization and other detrimental paramagnetic effects. AMUPol still yields the highest signal-to-noise improvement per unit time between the cross effect radicals due to faster polarization buildup (T1DNP = 4.3 s and 36 s for AMUPol and TEMTriPol-1, respectively). Overall, AMUPol results in 2.5× better sensitivity compared to TEMTriPol-1 in MAS DNP experiments performed below 6 K at 7 T. Trityl provides 6.0× more sensitivity than TEMTriPol-1 and 1.9× more than AMUPol at 6 K, thus yielding the greatest signal-to-noise per unit time among all three radicals. A DNP enhancement profile of TEMTriPol-1 recorded with a frequency-tunable custom-built gyrotron oscillator operating at 198 GHz is also included. It is determined that at 7 T below 6 K a microwave power level of 0.6 W incident on the sample is sufficient to saturate the cross effect mechanism using TEMTriPol-1, yet increasing the power level up to 5 W results in higher improvements in DNP sensitivity with AMUPol. These results indicate MAS DNP below 6 K will play a prominent role in ultra-sensitive NMR spectroscopy in the future.

Four millimeter spherical rotors spinning at 28 kHz with double-saddle coils for cross polarization NMR.

Spherical rotors in magic angle spinning (MAS) experiments have significant advantages over traditional cylindrical rotors including simplified spinning implementation, easy sample exchange, more efficient microwave coupling for dynamic nuclear polarization (DNP), and feasibility of downscaling to access higher spinning frequencies. Here, we implement spherical rotors with 4 mm outside diameter (o.d.) and demonstrate spinning >28 kHz using a single aperture for spinning gas. We show a modified stator geometry to improve fiber optic detection, increase NMR filling factor, and improve alignment for sample exchange and microwave irradiation. Higher NMR Rabi frequencies were obtained using smaller radiofrequency (RF) coils on small-diameter spherical rotors, compared to our previous implementation of MAS spheres with an o.d. of 9.5 mm. We report nutation fields of 110 kHz on 13C with 820 W of input power and 100 kHz on 1H with 800 W of input power. Proton decoupling fields of 78 kHz were applied over 20 ms of signal acquisition without any sign of arcing. Compared to our initial demonstration of a split coil for 9.5 mm spheres, this current implementation of a double-saddle coil inductor for 4 mm spheres not only intensifies the RF fields, but also improves RF homogeneity. We achieve an 810°/90° nutation intensity ratio of 0.84 at 300.197 MHz (1H). We also show electromagnetic simulations predicting a nearly 3-fold improvement in electron Rabi frequency of 0.99 MHz (with 4 mm spheres) compared to 0.38 MHz (with 3.2 mm cylinders), with 5 W of incident microwave power. Further improvements in magnetic resonance spin control are expected as RF inductors and microwave coupling are optimized for spherical rotors and scaled down to the micron scale.

Frequency-chirped dynamic nuclear polarization with magic angle spinning using a frequency-agile gyrotron.

We demonstrate that frequency-chirped dynamic nuclear polarization (DNP) with magic angle spinning (MAS) improves the enhancement of nuclear magnetic resonance (NMR) signal beyond that of continuous-wave (CW) DNP. Using a custom, frequency-agile gyrotron we implemented frequency-chirped DNP using the TEMTriPol-1 biradical, with MAS NMR at 7 T. Frequency-chirped microwaves yielded a DNP enhancement of 137, an increase of 19% compared to 115 recorded with CW. The chirps were 120 MHz-wide and centered over the trityl resonance, with 7 W microwave power incident on the sample (estimated 0.4 MHz electron spin Rabi frequency). We describe in detail the design and fabrication of the frequency-agile gyrotron used for frequency-chirped MAS DNP. Improvements to the interaction cavity and internal mode converter yielded efficient microwave generation and mode conversion, achieving >10 W output power over a 335 MHz bandwidth with >110 W peak power. Frequency-chirped DNP with MAS is expected to have a significant impact on the future of magnetic resonance.

Electron decoupling with cross polarization and dynamic nuclear polarization below 6 K.

Dynamic nuclear polarization (DNP) can improve nuclear magnetic resonance (NMR) sensitivity by orders of magnitude. Polarizing agents containing unpaired electrons required for DNP can broaden nuclear resonances in the presence of appreciable hyperfine couplings. Here we present the first cross polarization experiments implemented with electron decoupling, which attenuates detrimental hyperfine couplings. We also demonstrate magic angle spinning (MAS) DNP experiments below 6 K, producing unprecedented nuclear spin polarization in rotating solids. 13C correlation spectra were collected with MAS DNP below 6 K for the first time. Polarization build-up times with MAS DNP (T1DNP, 1H) of urea in a frozen glassy matrix below 6 K were measured for both the solid effect and the cross effect. Trityl radicals exhibit a T1DNP (1H) of 18.7 s and the T1DNP (1H) of samples doped with 20 mM AMUPol is only 1.3 s. MAS below 6 K with DNP and electron decoupling is an effective strategy to increase NMR signal-to-noise ratios per transient while retaining short polarization periods.

Pulsed Electron Decoupling and Strategies for Time Domain Dynamic Nuclear Polarization with Magic Angle Spinning.

Magic angle spinning (MAS) dynamic nuclear polarization (DNP) is widely used to increase nuclear magnetic resonance (NMR) signal intensity. Frequency-chirped microwaves yield superior control of electron spins and are expected to play a central role in the development of DNP MAS experiments. Time domain electron control with MAS has considerable promise to improve DNP performance at higher fields and temperatures. We have recently demonstrated that pulsed electron decoupling using frequency-chirped microwaves improves MAS DNP experiments by partially attenuating detrimental hyperfine interactions. The continued development of pulsed electron decoupling will enable a new suite of MAS DNP experiments that transfer polarization directly to observed spins. Time domain DNP transfers to nuclear spins in conjunction with pulsed electron decoupling is described as a viable avenue toward DNP-enhanced, high-resolution NMR spectroscopy over a range of temperatures from <6 to 320 K.

Magic angle spinning NMR below 6 K with a computational fluid dynamics analysis of fluid flow and temperature gradients.

We report magic angle spinning (MAS) up to 8.5 kHz with a sample temperature below 6 K using liquid helium as a variable temperature fluid. Cross polarization 13C NMR spectra exhibit exquisite sensitivity with a single transient. Remarkably, 1H saturation recovery experiments show a 1H T1 of 21 s with MAS below 6 K in the presence of trityl radicals in a glassy matrix. Leveraging the thermal spin polarization available at 4.2 K versus 298 K should result in 71 times higher signal intensity. Taking the 1H longitudinal relaxation into account, signal averaging times are therefore predicted to be expedited by a factor of >500. Computer assisted design (CAD) and finite element analysis were employed in both the design and diagnostic stages of this cryogenic MAS technology development. Computational fluid dynamics (CFD) models describing temperature gradients and fluid flow are presented. The CFD models bearing and drive gas maintained at 100 K, while a colder helium variable temperature fluid stream cools the center of a zirconia rotor. Results from the CFD were used to optimize the helium exhaust path and determine the sample temperature. This novel cryogenic experimental platform will be integrated with pulsed dynamic nuclear polarization and electron decoupling to interrogate biomolecular structure within intact human cells.

A versatile custom cryostat for dynamic nuclear polarization supports multiple cryogenic magic angle spinning transmission line probes.

Dynamic nuclear polarization (DNP) with cryogenic magic angle spinning (MAS) provides significant improvements in NMR sensitivity, yet presents unique technical challenges. Here we describe a custom cryostat and suite of NMR probes capable of manipulating nuclear spins with multi-resonant radiofrequency circuits, cryogenic spinning below 6 K, sample exchange, and microwave coupling for DNP. The corrugated waveguide and six transfer lines needed for DNP and cryogenic spinning functionality are coupled to the probe from the top of the magnet. Transfer lines are vacuum-jacketed and provide bearing and drive gas, variable temperature fluid, two exhaust pathways, and a sample ejection port. The cryostat thermally isolates the magnet bore, thereby protecting the magnet and increasing cryogen efficiency. This novel design supports cryogenic MAS-DNP performance over an array of probes without altering DNP functionality. We present three MAS probes (two supporting 3.2 mm rotors and one supporting 9.5 mm rotors) interfacing with the single cryostat. Mechanical details, transmission line radio frequency design, and performance of the cryostat and three probes are described.

Magic angle spinning spheres.

Magic angle spinning (MAS) is commonly used in nuclear magnetic resonance of solids to improve spectral resolution. Rather than using cylindrical rotors for MAS, we demonstrate that spherical rotors can be spun stably at the magic angle. Spherical rotors conserve valuable space in the probe head and simplify sample exchange and microwave coupling for dynamic nuclear polarization. In this current implementation of spherical rotors, a single gas stream provides bearing gas to reduce friction, drive propulsion to generate and maintain angular momentum, and variable temperature control for thermostating. Grooves are machined directly into zirconia spheres, thereby converting the rotor body into a robust turbine with high torque. We demonstrate that 9.5-mm-outside diameter spherical rotors can be spun at frequencies up to 4.6 kHz with N2(g) and 10.6 kHz with He(g). Angular stability of the spinning axis is demonstrated by observation of 79Br rotational echoes out to 10 ms from KBr packed within spherical rotors. Spinning frequency stability of ±1 Hz is achieved with resistive heating feedback control. A sample size of 36 µl can be accommodated in 9.5-mm-diameter spheres with a cylindrical hole machined along the spinning axis. We further show that spheres can be more extensively hollowed out to accommodate 161 µl of the sample, which provides superior signal-to-noise ratio compared to traditional 3.2-mm-diameter cylindrical rotors.

Frequency-agile gyrotron for electron decoupling and pulsed dynamic nuclear polarization.

We describe a frequency-agile gyrotron which can generate frequency-chirped microwave pulses. An arbitrary waveform generator (AWG) within the NMR spectrometer controls the microwave frequency, enabling synchronized pulsed control of both electron and nuclear spins. We demonstrate that the acceleration of emitted electrons, and thus the microwave frequency, can be quickly changed by varying the anode voltage. This strategy results in much faster frequency response than can be achieved by changing the potential of the electron emitter, and does not require a custom triode electron gun. The gyrotron frequency can be swept with a rate of 20 MHz/µs over a 670 MHz bandwidth in a static magnetic field. We have already implemented time-domain electron decoupling with dynamic nuclear polarization (DNP) magic angle spinning (MAS) with this device. In this contribution, we show frequency-swept DNP enhancement profiles recorded without changing the NMR magnet or probe. The profile of endofullerenes exhibits a DNP profile with a <10 MHz linewidth, indicating that the device also has sufficient frequency stability, and therefore phase stability, to implement pulsed DNP mechanisms such as the frequency-swept solid effect. We describe schematics of the mechanical and vacuum construction of the device which includes a novel flanged sapphire window assembly. Finally, we discuss how commercially available continuous-wave gyrotrons can potentially be converted into similar frequency-agile high-power microwave sources.

A flow-through, elevated-temperature and -pressure NMR apparatus for in-situ CO2 sequestration studies.

We report an apparatus for in-situ nuclear magnetic resonance (NMR) studies of chemical reactions of dissolved 13CO2 with minerals (rock or powder) under continuous flow. The operating range of the apparatus is 18-150°C and 1-140bar. A flow pump is used to circulate a CO2-water solution, with a heated mixing vessel where CO2 gas equilibrates with a water solution. The NMR probe is built around a strong zirconia ceramic vessel, with o-ring sealed connections; the mineral is contained inside. The horizontal orientation of the zirconia vessels allows use of a radio frequency (rf) solenoid for improved spin sensitivity.

A combined experimental setup for OP and ODNMR.

Instrumentation for optically-pumped and optically-detected nuclear magnetic resonance (OPNMR and ODNMR) has been developed and implemented as a single experimental apparatus to study semiconductors such as GaAs and CdTe. These two measurement schemes use many of the same components for experiments. Here we describe, in two parts, the apparatus that can record such measurements and give examples of representative data. In Part 1, the radio-frequency probe and low-temperature cryostat are described, including single-channel and two-channel static cryogenic probes that both incorporate a modified solenoid coil that permits better optical access. In Part 2, the optical bench is described in detail, which uses a set of experiments (magneto-photoluminescence, photoluminescence excitation, detection of polarized photoluminescence) as important input for ODNMR. We are able to portray a robust design that encompasses multiple measurement modalities, along with the ability to change many experimental parameters with ease.