Watch-sized 12 Tesla all-high-temperature-superconducting magnet.

We demonstrate the construction of 7 Tesla and 12 Tesla all high-temperature-superconducting (HTS) magnets, small enough to fit on your wrist. The size of the magnet reduces the cost of fabrication, decreases the fringe field to permit facile siting of magnets, and decreases the stored energy of high field magnets. These small HTS-based magnets are being developed for gyrotron microwave sources for use in high-field nuclear magnetic resonance applications. The 7 Tesla and 12 Tesla magnets employ a no-insulation winding technique and are cooled to 4.2 Kelvin in a liquid helium cryostat. The 7 Tesla magnet is a single pancake coil, made of only 9.4 m of HTS tape, with an inner diameter of 8 mm and an outer diameter of 24 mm. This magnet was charged up to 1168 Amperes, generating a field of 7.3 Tesla. The 12 Tesla magnet is comprised of two pancake coils (inner diameter of 10 mm and outer diameter of 27 mm) connected in series. This magnet reached its maximum field at a current of 850 Amperes.

Data-Driven Prediction of Molecular Biotransformations in Food Fermentation.

Fermentation products, together with food components, determine the sense, nutrition, and safety of fermented foods. Traditional methods of fermentation product identification are time-consuming and cumbersome, which cannot meet the increasing need for the identification of the extensive bioactive metabolites produced during food fermentation. Hence, we propose a data-driven integrated platform (FFExplorer, http://www.rxnfinder.org/ffexplorer/) based on machine learning and data on 2,192,862 microbial sequence-encoded enzymes for computational prediction of fermentation products. Using FFExplorer, we explained the mechanism behind the disappearance of spicy taste during pepper fermentation and evaluated the detoxification effects of microbial fermentation for common food contaminants. FFExplorer will provide a valuable reference for inferring bioactive "dark matter" in fermented foods and exploring the application potential of microorganisms.

Data-Driven Elucidation of Flavor Chemistry.

Flavor molecules are commonly used in the food industry to enhance product quality and consumer experiences but are associated with potential human health risks, highlighting the need for safer alternatives. To address these health-associated challenges and promote reasonable application, several databases for flavor molecules have been constructed. However, no existing studies have comprehensively summarized these data resources according to quality, focused fields, and potential gaps. Here, we systematically summarized 25 flavor molecule databases published within the last 20 years and revealed that data inaccessibility, untimely updates, and nonstandard flavor descriptions are the main limitations of current studies. We examined the development of computational approaches (e.g., machine learning and molecular simulation) for the identification of novel flavor molecules and discussed their major challenges regarding throughput, model interpretability, and the lack of gold-standard data sets for equitable model evaluation. Additionally, we discussed future strategies for the mining and designing of novel flavor molecules based on multi-omics and artificial intelligence to provide a new foundation for flavor science research.

Improving the sensitivity of MAS spheres using a 9.5 mm spherical shell with 219 µL sample volume spinning in a spherical solenoid coil.

Spherical rotors in magic angle spinning (MAS) nuclear magnetic resonance (NMR) experiments have potential advantages relative to cylindrical rotors in terms of ease of fabrication, low risk of rotor crash, easy sample exchange, and better microwave access. However, one major disadvantage so far of spherical rotors is poor NMR filling factor due to the small sample volume and large cylindrical radiofrequency (RF) coil. Here we present a novel NMR coil geometry in the form of a spherical coil. The spherical coil best fits the spherical sample to maximize sensitivity, while also providing excellent RF homogeneity. We further improve NMR sensitivity by employing a spherical shell as the rotor, thereby maximizing sample volume (219 µL in this case of 9.5 mm outer diameter spheres). The spinning gas is supplied by a 3D-printed ring stator external to the coil, thereby introducing a simplified form of MAS stators. In this apparatus, the RF field generated along the coil axis is perpendicular to the external magnetic field, regardless of rotor orientation. We observe a linear increase in sensitivity with increasing sample volume. We also simulate the RF performance of spherical and cylindrical solenoid coils with constant or variable pitch for spherical and cylindrical rotors, respectively. The simulation results show that spherical solenoid coils generate comparable B1 field intensities but have better homogeneity than cylindrical solenoid coils do.

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.

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.

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.

Perspectives on microwave coupling into cylindrical and spherical rotors with dielectric lenses for magic angle spinning dynamic nuclear polarization.

Continuous wave dynamic nuclear polarization (DNP) increases the sensitivity of NMR, yet intense microwave fields are required to transition magic angle spinning (MAS) DNP to the time domain. Here we describe and analyze Teflon lenses for cylindrical and spherical MAS rotors that focus microwave power and increase the electron Rabi frequency, ν1s. Using a commercial simulation package, we solve the Maxwell equations and determine the propagation and focusing of millimeter waves (198 GHz). We then calculate the microwave intensity in a time-independent fashion to compute the ν1s. With a nominal microwave power input of 5 W, the average ν1s is 0.38 MHz within a 22 µL sample volume in a 3.2 mm outer diameter (OD) cylindrical rotor without a Teflon lens. Decreasing the sample volume to 3 µL and focusing the microwave beam with a Teflon lens increases the ν1s to 1.5 MHz. Microwave polarization and intensity perturbations associated with diffraction through the radiofrequency coil, losses from penetration through the rotor wall, and mechanical limitations of the separation between the lens and sample are significant challenges to improving microwave coupling in MAS DNP instrumentation. To overcome these issues, we introduce a novel focusing strategy using dielectric microwave lenses installed within spinning rotors. One such 9.5 mm OD cylindrical rotor assembly implements a Teflon focusing lens to increase the ν1s to 2.7 MHz within a 2 µL sample. Further, to access high spinning frequencies while also increasing ν1s, we analyze microwave coupling into MAS spheres. For 9.5 mm OD spherical rotors, we compute a ν1s of 0.36 MHz within a sample volume of 161 µL, and 2.5 MHz within a 3 µL sample placed at the focal point of a novel double lens insert. We conclude with an analysis and discussion of sub-millimeter diamond spherical rotors for time domain DNP at spinning frequencies >100 kHz. Sub-millimeter spherical rotors better overlap a tightly focused microwave beam, resulting in a ν1s of 2.2 MHz. Lastly, we propose that sub-millimeter dielectric spherical microwave resonators will provide a means to substantially improve electron spin control 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.

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.

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.

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.

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%.

Post-synthetic modification of tryptophan containing peptides via NIS mediation.

A new efficient method was developed to provide modified tryptophan peptides through NIS (N-iodosuccinimide) mediated N2-selective coupling of a Trp unit with 1,2,3-triazoles, of which, the preliminary spectral properties were also studied.