Plasmonic Metasurface Resonators to Enhance Terahertz Magnetic Fields for High-Frequency Electron Paramagnetic Resonance.

Nanoscale magnetic systems play a decisive role in areas ranging from biology to spintronics. Although, in principle, THz electron paramagnetic resonance (EPR) provides high-resolution access to their properties, lack of sensitivity has precluded realizing this potential. To resolve this issue, the principle of plasmonic enhancement of electromagnetic fields that is used in electric dipole spectroscopies with great success is exploited, and a new type of resonators for the enhancement of THz magnetic fields in a microscopic volume is proposed. A resonator composed of an array of diabolo antennas with a back-reflecting mirror is designed and fabricated. Simulations and THz EPR measurements demonstrate a 30-fold signal increase for thin film samples. This enhancement factor increases to a theoretical value of 7500 for samples confined to the active region of the antennas. These findings open the door to the elucidation of fundamental processes in nanoscale samples, including junctions in spintronic devices or biological membranes.

X-band TE101 rectangular aperture cavity for in vivo EPR tooth dosimetry after radiation emergency.

The TE101 mode rectangle EPR cavity was newly developed to achieve X-band in vivo EPR tooth dosimetry for the rescue of nuclear emergency. An aperture for sample detection was opened on the cavity's surface. Its characteristics were evaluated by measuring DPPH and intact human incisor samples. Remarkable radiation induced signal from EPR spectrum of 1Gy-8Gy irradiated teeth was observed. In vivo measurements of rat was performed to verify its application for in vivo tooth dosimetry.

Protocol for resolving enzyme orientation and dynamics in advanced porous materials via SDSL-EPR.

Enzyme encapsulation in metal-organic frameworks (MOFs)/covalent-organic frameworks (COFs) provides advancement in biocatalysis, yet the structural basis underlying the catalytic performance is challenging to probe. Here, we present an effective protocol to determine the orientation and dynamics of enzymes in MOFs/COFs using site-directed spin labeling and electron paramagnetic resonance spectroscopy. The protocol is demonstrated using lysozyme and can be generalized to other enzymes. For complete information on the generation and use of this protocol, please refer to Pan et al. (2021a).

Dual channel EPR excitation coil array for Overhauser-enhanced MRI.

This article describes the development of a two-channel surface coil array for Overhauser-enhanced magnetic resonance imaging (OMRI) with the aim of extending the visualization area. The surface coil array consists of two independent surface coil resonators and PIN-diode switches. We utilized the PIN-diode switch to decouple the surface coils. OMRI measurement using a surface coil array was sequentially performed by switching the channels. To evaluate the effectiveness of the surface coil array, we demonstrated OMRI measurements using a phantom filled with nitroxide solution. In addition, in vivo OMRI imaging with a mouse was performed to demonstrate the applicability of our surface coil array to in vivo measurements. As a result, the visualization area obtained with our surface coil array was extended approximately 2-fold compared to the conventional single surface coil. Furthermore, we showed that in vivo imaging with the surface coil array was possible. These results indicate that the surface coil array could enhance the applicability of OMRI imaging.

Ultra-low frequency EPR using longitudinal detection and fictitious-field modulation.

When viewed in a rotating frame of reference, a transverse-plane radiofrequency (RF) field manifests as a longitudinal field component called the fictitious field. By modulating the RF field and thus the fictitious field, detectable longitudinal magnetization patterns have previously been shown to be measurable. By combining fictitious-field modulation and longitudinal detection, here we demonstrate EPR spectroscopy and one-dimensional imaging in a custom-built longitudinal detection system operating at an ultra-low frequency (24 MHz) for detecting electron spins with short (~nanoseconds) relaxation times. Simultaneous transmit and receive with low transmitter leakage level (~80 dB isolation) is also demonstrated.

Effective coupling of rapid freeze-quench to high-frequency electron paramagnetic resonance.

We report an easy, efficient and reproducible way to prepare Rapid-Freeze-Quench samples in sub-millimeter capillaries and load these into the probe head of a 275 GHz Electron Paramagnetic Resonance spectrometer. Kinetic data obtained for the binding reaction of azide to myoglobin demonstrate the feasibility of the method for high-frequency EPR. Experiments on the same samples at 9.5 GHz show that only a single series of Rapid-Freeze-Quench samples is required for studies at multiple microwave frequencies.

Rutile dielectric loop-gap resonator for X-band EPR spectroscopy of small aqueous samples.

The performance of a metallic microwave resonator that contains a dielectric depends on the separation between metallic and dielectric surfaces, which affects radio frequency currents, evanescent waves, and polarization charges. The problem has previously been discussed for an X-band TE011 cylindrical cavity resonator that contains an axial dielectric tube (Hyde and Mett, 2017). Here, a short rutile dielectric tube inserted into a loop-gap resonator (LGR) at X-band, which is called a dielectric LGR (dLGR), is considered. The theory is developed and experimental results are presented. It was found that a central sample loop surrounded by four "flux-return" loops (i.e., 5-loop-4-gap) is preferable to a 3-loop-2-gap configuration. For sufficiently small samples (less than 1 µL), a rutile dLGR is preferred relative to an LGR both at constant Λ (B1/Pl) and at constant incident power. Introduction of LGR technology to X-band EPR was a significant advance for site-directed spin labeling because of small sample size and high Λ. The rutile dLGR introduced in this work offers further extension to samples that can be as small as 50  nL when using typical EPR acquisition times.

Quantum diamond spectrometer for nanoscale NMR and ESR spectroscopy.

Nitrogen-vacancy (NV) quantum defects in diamond are sensitive detectors of magnetic fields. Owing to their atomic size and optical readout capability, they have been used for magnetic resonance spectroscopy of nanoscale samples on diamond surfaces. Here, we present a protocol for fabricating NV diamond chips and for constructing and operating a simple, low-cost 'quantum diamond spectrometer' for performing NMR and electron spin resonance (ESR) spectroscopy in nanoscale volumes. The instrument is based on a commercially available diamond chip, into which an NV ensemble is ion-implanted at a depth of ~10 nm below the diamond surface. The spectrometer operates at low magnetic fields (~300 G) and requires standard optical and microwave (MW) components for NV spin preparation, manipulation, and readout. We demonstrate the utility of this device for nanoscale proton and fluorine NMR spectroscopy, as well as for the detection of transition metals via relaxometry. We estimate that the full protocol requires 2-3 months to implement, depending on the availability of equipment, diamond substrates, and user experience.

Modular imaging system: Rapid scan EPR at 800 MHz.

An electron paramagnetic resonance (EPR) imaging system has been custom built for use in pre-clinical and, potentially, clinical studies. Commercial standalone modules have been used in the design that are MATLAB-controlled. The imaging system combines digital and analog technologies. It was designed to achieve maximum flexibility and versatility and to perform standard and novel user-defined experiments. This design goal is achieved by frequency mixing of an arbitrary waveform generator (AWG) output at the intermediate frequency (IF) with a constant source frequency (SF). Low noise SF at 250, 750, and 1000 MHz are available in the system. A wide range of frequencies from near-baseband to L-band can be generated as a result. Two-stage downconversion at the signal detection side is implemented that enables multi-frequency EPR capability. In the first stage, the signal frequency is converted to IF. A novel AWG-enabled digital auto-frequency control method that operates at IF is described that is used for automatic resonator tuning. Quadrature baseband EPR signal is generated in the second downconversion step. The semi-digital approach of mixing low-noise frequency sources with an AWG permits generation of arbitrary excitation patterns that include but are not limited to frequency sweeps for resonator tuning and matching, continuous-wave, and pulse sequences. Presented in this paper is the demonstration of rapid scan (RS) EPR imaging implemented at 800 MHz. Generation of stable magnetic scan waveforms is critical for the RS method. A digital automatic scan control (DASC) system was developed for sinusoidal magnetic field scans. DASC permits tight control of both amplitude and phase of the scans. A surface loop resonator was developed using 3D printing technology. RS EPR imaging system was validated using sample phantoms. In vivo imaging of a breast cancer mouse model is demonstrated.

Development of an L-band rapid scan EPR digital console.

The development of a digital console for in-vivo rapid scan electron paramagnetic resonance (RS-EPR) spectroscopy and imaging is described in detail. The console was build using field programmable gate array (FGPA) technology that permits real-time control of the resonator and scanning magnetic fields during the measurements. Automatic resonator tuning and matching are achieved by implementing a digital feedback control system and using voltage-tunable capacitors. A band-pass subsampling method is used to directly digitize EPR signals at the carrier frequencies of about 1.2 GHz. The magnetic field scan waveforms, excitation EPR frequency, and sampling clock are all internally synchronized. Full-cycle RS-EPR signals are accumulated in the FPGA in real time without any time gaps. The result is the elimination of the re-arm time, during which data are not acquired. The proposed design in this manuscript has a small footprint and is relatively low cost. The FPGA-based RS-EPR system was tested using standard LiNc-BuO and tempone-d16 samples. The RS-EPR linewidth of the LiNc-BuO sample was consistent with an independent pulsed EPR measurement.

INITIAL TL/OSL/EPR CONSIDERATIONS FOR COMMERCIAL DIATOMACEOUS EARTH IN RETROSPECTIVE DOSIMETRY AND DATING.

Diatomaceous earth is found in various locations around the planet. It is caused by the deposited exoskeleton material formed by the death of large concentrated populations of diatoms. The exoskeleton is effectively pure silicate and as such becomes a prospective material for retrospective dosimetry and dating. This work investigated the thermoluminescence (TL) and optically stimulated luminescence properties of commercially obtained diatomaceous earth. The material was not found to have useful dosimetric properties with conventional TL methodologies but did provide large dose estimates using the Single Aliquot Regeneration technique on some subset samples. These findings for organic silicate did suggest some mechanisms explaining the sensitization process in geological silicate materials utilized in dosimetry and dating. Electron paramagnetic resonance was identified as a potential future method for evaluating this material as it revealed unique signal components not found in igneous or commercially produced silicates.

CHARACTERIZATION OF A LITHIUM FORMATE EPR-DOSIMETRY SYSTEM FOR PROTON RADIATION THERAPY.

The specific aim for the characterization of the lithium formate dosimetry system is to determine response and stability in a proton beam. The long-term goal for this investigation is an audit system for proton therapy like the end-to-end dose determinations performed for radiotherapy with photons. For a 150-MeV proton beam, the dose response was found to be linear in the dose interval 0-8.8 Gy. The accuracy of dose reconstruction was controlled in a blind test, in which the dose of 6.63 Gy was measured in samples irradiated with a real dose of 6.70 Gy. The stability was determined by irradiations of sets of four dosimeters every week during 1 month and analyzed at the same day thereafter. The fitting of the fading curve was done with a second-order polynomial resulting in a 6.6% lower value compared to the reference after 31 d.

Uniform Field Resonators for EPR Spectroscopy: A Review.

Cavity resonators are often used for electron paramagnetic resonance (EPR). Rectangular TE102 and cylindrical TE011 are common modes at X-band even though the field varies cosinusoidally along the Z-axis. The authors found a way to create a uniform field (UF) in these modes. A length of waveguide at cut-off was introduced for the sample region, and tailored end sections were developed that supported the microwave resonant mode. This work is reviewed here. The radio frequency (RF) magnetic field in loop-gap resonators (LGR) at X-band is uniform along the Z-axis of the sample, which is a benefit of LGR technology. The LGR is a preferred structure for EPR of small samples. At Q-band and W-band, the LGR often exhibits nonuniformity along the Z-axis. Methods to trim out this nonuniformity, which are closely related to the methods used for UF cavity resonators, are reviewed. In addition, two transmission lines that are new to EPR, dielectric tube waveguide and circular ridge waveguide, were recently used in UF cavity designs that are reviewed. A further benefit of UF resonators is that cuvettes for aqueous samples can be optimum in cross section along the full sample axis, which improves quantification in EPR spectroscopy of biological samples.

Electron Paramagnetic Resonance Spectroscopy of Metalloproteins.

Electron paramagnetic resonance (EPR) is a spectroscopic technique that is sensitive to the presence of unpaired electrons and, therefore, is a powerful tool for the study of proteins containing complex metallocofactors. When a magnetic field is applied to a transition metal-containing system with unpaired electrons and the sample is irradiated with microwaves, a spin transition can be observed. Through detailed analysis of the resulting EPR spectrum, one can extract parameters that can provide information about the electronic environment of the unpaired electrons found on the metal centers. Here, a basic introduction to the theory of EPR and the instrumentation is presented along with procedures for obtaining EPR spectra of sensitive metalloprotein species.

EPR Lineshape Analysis to Investigate the SNARE Folding Intermediates.

SNARE complex formation, which is believed to drive intracellular membrane fusion, transits through multiple conformational states along the membrane fusion pathway. The SNARE intermediates are biologically important because they serve as targets for fusion regulators and clostridial neurotoxins. Spin-labeling EPR has contributed significantly to the understanding of the structures and the dynamics of SNARE intermediates. In particular, the EPR lineshape analysis, which is highly sensitive to protein conformational changes such as the local coil-to-helix transition, has revealed the sequential compacting steps leading to formation of the highly stable four-helix bundle.

Towards reduction of SAR in scaling up in vivo pulsed EPR imaging to larger objects.

An excessive RF power requirement is one of the main obstacles in the clinical translation of EPR imaging. The radio frequency (RF) pulses used in EPR imaging to excite electron spins must be very short to match their fast relaxation. With traditional pulse schemes and ninety degree flip angles, this can lead to either unsafe specific absorption rate (SAR) levels or unfeasibly long repetition times. In spectroscopy experiments, it has been shown that stochastic excitation and correlation detection can reduce the power while maintaining sensitivity but have yet to be applied to imaging experiments. Stochastic excitation is implemented using a pseudo-random phase modulation of the input stimulus. Using a crossed coil resonator assembly comprised of an outer saddle coil and an inner surface coil, it was possible to obtain a minimum isolation of ∼50 dB across a 12 MHz bandwidth. An incident peak RF power of 5 mW was used to excite the system. The low background signal obtained from this resonator allowed us to generate images with 32 dB (>1000:1) signal-to-noise ratio (SNR) while exciting with a traditional pulse sequence in a phantom containing the solid paramagnetic probe NMP-TCNQ (N-methyl pyridinium tetracyanoquinodimethane). Using two different stochastic excitation schemes, we were able to achieve a greater than 4-fold increase in SNR at the same peak power and number of averages, compared to single pulse excitation. This procedure allowed imaging at significantly lower RF power levels than used in conventional EPR imaging system configurations. Similar techniques may enable clinical applications for EPR imaging by facilitating the use of larger RF coils while maintaining a safe SAR level.

Development of a novel mouth model as an alternative tool to test the effectiveness of an in vivo EPR dosimetry system.

In a large-scale radiation event, thousands may be exposed to unknown amounts of radiation, some of which may be life-threatening without immediate attention. In such situations, a method to quickly and reliably estimate dose would help medical responders triage victims to receive life-saving care. We developed such a method using electron paramagnetic resonance (EPR) to make in vivo measurements of the maxillary incisors. This report provides evidence that the use of in vitro studies can provide data that are fully representative of the measurements made in vivo. This is necessary because, in order to systematically test and improve the reliability and accuracy of the dose estimates made with our EPR dosimetry system, it is important to conduct controlled studies in vitro using irradiated human teeth. Therefore, it is imperative to validate whether our in vitro models adequately simulate the measurements made in vivo, which are intended to help guide decisions on triage after a radiation event. Using a healthy volunteer with a dentition gap that allows using a partial denture, human teeth were serially irradiated in vitro and then, using a partial denture, placed in the volunteer's mouth for measurements. We compared dose estimates made using in vivo measurements made in the volunteer's mouth to measurements made on the same teeth in our complex mouth model that simulates electromagnetic and anatomic properties of the mouth. Our results demonstrate that this mouth model can be used in in vitro studies to develop the system because these measurements appropriately model in vivo conditions.

Electron-Spin-Resonance Dipstick.

Electron spin resonance (ESR) is a powerful analytical technique used for the detection, quantification, and characterization of paramagnetic species ranging from stable organic free radicals and defects in crystals to gaseous oxygen. Traditionally, ESR requires the use of complex instrumentation, including a large magnet and a microwave resonator in which the sample is placed. Here, we present an alternative to the existing approach by inverting the typical measurement topology, namely placing the ESR magnet and resonator inside the sample rather than the other way around. This new development relies on a novel self-contained ESR sensor with a diameter of just 2 mm and length of 3.6 mm, which includes both a small permanent magnet assembly and a tiny (∼1 mm in size) resonator for spin excitation and detection at a frequency of ∼2.6 GHz. The spin sensitivity of the sensor has been measured to be ∼1011 spins/√Hz, and its concentration sensitivity is ∼0.1 mM, using reference samples with a measured volume of just ∼10 nL. Our new approach can be applied for monitoring the partial pressure of oxygen in vitro and in vivo through its paramagnetic interaction with another stable radical, as well as for simple online quantitative inspection of free radicals generated in reaction vessels and electrochemical cells via chemical processes.

A combined positron emission tomography (PET)-electron paramagnetic resonance imaging (EPRI) system: initial evaluation of a prototype scanner.

The advent of hybrid scanners, combining complementary modalities, has revolutionized the application of advanced imaging technology to clinical practice and biomedical research. In this project, we investigated the melding of two complementary, functional imaging methods: positron emission tomography (PET) and electron paramagnetic resonance imaging (EPRI). PET radiotracers can provide important information about cellular parameters, such as glucose metabolism. While EPR probes can provide assessment of tissue microenvironment, measuring oxygenation and pH, for example. Therefore, a combined PET/EPRI scanner promises to provide new insights not attainable with current imagers by simultaneous acquisition of multiple components of tissue microenvironments. To explore the simultaneous acquisition of PET and EPR images, a prototype system was created by combining two existing scanners. Specifically, a silicon photomultiplier (SiPM)-based PET scanner ring designed as a portable scanner was combined with an EPRI scanner designed for the imaging of small animals. The ability of the system to obtain simultaneous images was assessed with a small phantom consisting of four cylinders containing both a PET tracer and EPR spin probe. The resulting images demonstrated the ability to obtain contemporaneous PET and EPR images without cross-modality interference. Given the promising results from this initial investigation, the next step in this project is the construction of the next generation pre-clinical PET/EPRI scanner for multi-parametric assessment of physiologically-important parameters of tissue microenvironments.

Wireless implantable coil with parametric amplification for in vivo electron paramagnetic resonance oximetric applications.

PURPOSE: To develop an implantable wireless coil with parametric amplification capabilities for time-domain electron paramagnetic resonance (EPR) spectroscopy operating at 300 MHz. METHODS: The wireless coil and lithium phthalocyanine (LiPc), a solid paramagnetic probe, were each embedded individually in a biocompatible polymer polydimethoxysiloxane (PDMS). EPR signals from the LiPc embedded in PDMS (LiPc/PDMS) were generated by a transmit-receive surface coil tuned to 300 MHz. Parametric amplification was configured with an external pumping coil tuned to 600 MHz and placed between the surface coil resonator and the wireless coil. RESULTS: Phantom studies showed significant enhancement in signal to noise using the pumping coil. However, no influence of the pumping coil on the oxygen-dependent EPR spectral linewidth of LiPc/PDMS was observed, suggesting the validity of parametric amplification of EPR signals for oximetry by implantation of the encapsulated wireless coil and LiPc/PDMS in deep regions of live objects. In vivo studies demonstrate the feasibility of this approach to longitudinally monitor tissue pO2 in vivo and also monitor acute changes in response to pharmacologic challenges. The encapsulated wireless coil and LiPc/PDMS engendered no host immune response when implanted for ∼3 weeks and were found to be well tolerated. CONCLUSIONS: This approach may find applications for monitoring tissue oxygenation to better understand the pathophysiology associated with wound healing, organ transplantation, and ischemic diseases.