Over the last 50 years, EPR oximetry has been established as the gold standard for absolute quantitative measurement and imaging of oxygen partial pressure in the biomedical setting. In this review, the main developments in EPR oximetry through its history are discussed. The first oxygen measurement with EPR was reported approximately 50 years ago, in 1975. Since then, a wide variety of probes have been developed for various biomedical applications including preclinical and clinical study. These probes can be categorized into soluble probes including nitroxides and trityls for volumetric imaging, and solid-state probes, for long-term point measurements. Oximetry measurements range in size from a single cell to whole body rabbit imaging. The applications of preclinical EPR oximetry are still growing in scope, and include cancer, myocardial infarction, stroke, and wound healing. First studies in clinical oximetry appear promising, with discrete measurement of oxygen using injected ink or implanted OxyChip probes.

Electron paramagnetic resonance (EPR) has been established as a unique and reliable method for quantitative in vivo oximetry applicable to a variety of preclinical and clinical studies. A recent clinical study using EPR oximetry with OxyChip from our laboratory demonstrated the feasibility of tumor oxygen measurements in cancer patients (Schaner, et al. Front. Oncol. 2021). During this study, the need to improve oxygen measurement capability in tumors at depths greater than 10 mm became apparent. This prompted us to develop new designs of resonators (RF coils) with enhanced sensitivity for measuring deep-tissue oxygen levels. In this manuscript, we report the development of a new cylindrical surface dielectric resonator (c-SDR) designed with a ceramic dielectric material for substantially enhanced sensitivity and capability for deep-tissue oximetry. The c-SDR was constructed with a cylindrical dielectric material (ϕ 27.2 × 22.2 mm; ε = 160), 6-segmented coupling loop and copper shield to provide an active surface (aperture) of 25 mm with an operating frequency of 1.16 GHz (L-band) and an unloaded Q 600. The resonator could detect OxyChip (ϕ 0.6 × 5 mm) at a surface-to-sample depth of 50 mm in water or 30 mm in a tissue-emulating phantom with a signal-to-noise ratio of 5. Further evaluations of the c-SDR using OxyChip demonstrated its capability for oxygen measurements at depths of 27 mm for 1

This research report describes a novel surface dielectric resonator (SDR) with a flexible connector for in vivo electron paramagnetic resonance (EPR) spectroscopy. Contrary to the conventional cavity or surface loop-gap resonators, the newly developed SDR is constructed from a ceramic dielectric material, and it is tuned to operate at the L-band frequency band (1.15 GHz) in continuous-wave mode. The SDR is designed to be critically coupled and capable of working with both very lossy samples, such as biological tissues, and non-lossy materials. The SDR was characterized using electromagnetic field simulations, assessed for sensitivity with a B 1 field-perturbation method, and validated with tissue phantoms using EPR measurements. The results showed remarkably higher sensitivity in lossy tissue phantoms than the previously reported multisegment surface-loop resonators. The new SDR can provide potential new insights for advancements in the application of in vivo EPR spectroscopy for biological measurements, including clinical oximetry.

EPR spectrometers (and various sub-systems) have been designed and constructed to facilitate in vivo measurements with human subjects for dosimetry and oximetry. Most applications are primarily focused on surface tissue measurements; however, oximetric measurements utilizing implantable devices are also discussed. Given various specifications and considerations across these two primary applications, several embodiments and configurations of the associated sub-systems to the spectrometer have been realized and implemented. These embodiments and configurations have been developed and tested with a focus on the end-use and the end-users. This includes acknowledgement of the challenges and needs for making measurements in unanesthetized human patients, the nature of the data that are most likely to be useful for clinical decision-making, and the person who will be making the actual measurements. Significant developments in spectrometer automation and features to ensure the quality of recorded data are featured.

There is a strong need to enable accurate and convenient oxygen measurements in vivo for human subjects to improve treatments for cancer, peripheral vascular disease, and other diseases where tissue oxygen levels have a significant impact. While EPR spectroscopy has the potential to do this effectively, the full exploitation of these capabilities requires optimization of resonators for use with human subjects. Patient motion, and its effects on resonator coupling and positioning relative to the implanted oximetry probe, is a major source of noise and artifacts. Additionally, optimization of detection sensitivity to enable measurements from tissues at depths of several centimeters with clinically practical acquisition times is needed. To meet these needs, surface resonators with high sensitivity and flexible cables that allow the detection loop to be conveniently attached to the skin surface were developed for use with low frequency (L-Band, 1.15 GHz) continuous wave EPR. These resonators include a multi-segment sensing loop with a common capacitance. The light-weight segmented sensing loops, with diameters of 10–20 mm, can be connected to a commercial topical fixation applicator to conveniently and securely position them on patients’ skin surfaces. It was shown that a resonator of 20 mm in diameter makes it possible to obtain adequate EPR signals in tissue phantoms up to a depth of 20 mm with ~ 2 min of signal averaging. This novel lightweight resonator design, with sensitive multi-segment design and flexible cable with skin surface attachment, significantly reduced the impacts of subject motion enabling reliable EPR oximetry measurements in human subjects.

Objective The overall objective of this clinical study was to validate an implantable oxygen sensor, called the ‘OxyChip’, as a clinically feasible technology that would allow individualized tumor-oxygen assessments in cancer patients prior to and during hypoxia-modification interventions such as hyperoxygen breathing. Methods Patients with any solid tumor at ≤3-cm depth from the skin-surface scheduled to undergo surgical resection (with or without neoadjuvant therapy) were considered eligible for the study. The OxyChip was implanted in the tumor and subsequently removed during standard-of-care surgery. Partial pressure of oxygen (pO 2 ) at the implant location was assessed using electron paramagnetic resonance (EPR) oximetry. Results Twenty-three cancer patients underwent OxyChip implantation in their tumors. Six patients received neoadjuvant therapy while the OxyChip was implanted. Median implant duration was 30 days (range 4–128 days). Forty-five successful oxygen measurements were made in 15 patients. Baseline pO 2 values were variable with overall median 15.7 mmHg (range 0.6–73.1 mmHg); 33% of the values were below 10 mmHg. After hyperoxygenation, the overall median pO 2 was 31.8 mmHg (range 1.5–144.6 mmHg). In 83% of the measurements, there was a statistically significant (p ≤ 0.05) response to hyperoxygenation. Conclusions Measurement of baseline pO 2 and response to hyperoxygenation using EPR oximetry with the OxyChip is clinically feasible in a variety of tumor types. Tumor oxygen at baseline differed significantly among patients. Although most tumors responded to a hyperoxygenation intervention, some were non-responders. These data demonstrated the need for individualized assessment of tumor oxygenation in the context of planned hyperoxygenation interventions to optimize clinical outcomes.

Purpose To develop a novel resonator for high‐quality fast scan electron paramagnetic resonance (EPR) and EPR/NMR co‐imaging of the head and brain of mice at 1.25 GHz. Methods Resonator dimensions were scaled to fit the mouse head with maximum filling factor. A single‐loop 6‐gap resonator of 20 mm diameter and 20 mm length was constructed. High resonator stability was achieved utilizing a fixed position double coupling loop. Symmetrical mutually inverted connections rendered it insensitive to field modulation and fast scan. Coupling adjustment was provided by a parallel‐connected variable capacitor located at the feeding line at λ/4 distance. To minimize radiation loss, the shield around the resonator was supplemented with a planar conductive disc that focuses return magnetic flux. Results Coupling of the resonator loaded with the mouse head was efficient and easy. This resonator enabled high‐quality in vivo 3D EPR imaging of the mouse head following intravenous infusion of nitroxide probes. With this resonator and rapid scan EPR system, 4 ms scans were acquired in forward and reverse directions so that images with 2‐scan 3,136 projections were acquired in 25 s. Head images were achieved with resolutions of 0.4 mm, enabling visualization of probe localization and uptake across the blood–brain barrier. Conclusions This resonator design provides good sensitivity, high stability, and B 1 field homogeneity for in vivo fast scan EPR of the mouse head and brain, enabling faster measurements and higher resolution imaging of probe uptake, localization, and metabolism than previously possible.

In pulsed electron paramagnetic resonance (EPR), free-induction decay (FID) or spin echo (SE) signals of unpaired electrons are recorded in the time-domain. In both methods, electromagnetic waves play an important role in the detection of unpaired electrons in EPR spectroscopy. The resonator generates and senses electromagnetic waves and therefore serves as a critical interface between unpaired electrons and the transmit/receive systems of an EPR spectrometer. Since a resonator is a sensitive electrical circuit that can amplify voltages and currents when the electrical circuit of the resonator is on resonance, the resonator is an essential component for EPR detection in continuous wave and pulsed EPR. Without the resonator, EPR signals cannot be detected with sufficient sensitivity. In this chapter, the basics of resonators and some examples of resonators used in preclinical studies with small animals and human subjects are explained.

PurposeIn continuous wave EPR imaging, the acquisition of high-quality images was previously limited by the requisite long acquisition times of each image projection that was typically greater than 1 second. To accelerate the process of image acquisition facilitating greater numbers of projections and higher image resolution, instrumentation was developed to greatly accelerate the magnetic field scan that is used to obtain each EPR image projection. MethodsA low-inductance solenoidal coil for field scanning was used along with a spherical solenoid air core magnet, and scans were driven by triangular symmetric waves, allowing forward and reverse spectrum acquisition as rapid as 3.8 ms. The uniform distribution of projections was used to optimize the contribution of projections for 3D image reconstruction. ResultsUsing this fast-scan EPR system, high-quality EPR images of phantoms and perfused rat hearts were performed using trityl or nanoparticulate LiNcBuO (lithium octa-n-butoxy-substituted naphthalocyanine) probes with fast-scan EPR imaging at L-band, achieving spatial resolutions of up to 250 micrometers in 1 minute. ConclusionFast-scan EPR imaging can greatly facilitate the efficient and precise mapping of the spatial distribution of free radical and other paramagnetic probes in living systems.

Clinical interventions to mitigate the impact of hypoxia on cancer control outcomes have been hampered by an inability to assess patient specific tumor oxygenation. This study aimed to validate a novel implantable oxygen sensor (OxyChip) as a clinically feasible technology that will allow for individualized tissue oxygen assessment prior to and during hypoxia modification therapies. After Oxychip implantation the absolute partial pressure of oxygen (pO2) around the sensor was assessed using electron paramagnetic resonance (EPR) oximetry. Here, we present updated data examining tissue oxygen in a variety of tumors prior to and during cancer therapies. Patients with a tumor < 3 cm depth from the skin surface slated to receive surgical resection (with or without neoadjuvant therapy) were eligible. Patients were implanted with an Oxychip that was subsequently removed during surgery. Tissue oxygenation was assessed using a clinical EPR scanner and surface loop detector; at each oximetry session measurements were taken with a patient breathing room air for 10 min (baseline) followed by 10 min of breathing a hyperoxygenated gas mixture. Differences in absolute pO2 were assessed via a two-tailed unpaired Students t-test. Twenty three patients were implanted; the majority had skin (47%) or breast (35%) cancers. Six underwent neoadjuvant therapy. Median implant duration was 20 days (range 3 – 46) in patients who received surgery alone and 130 days (range 123 – 137) in patients receiving neoadjuvant chemotherapy. The majority of OxyChips were found within tumor after surgery. Baseline pO2 was < 10 mmHg in 17 of 40 measurements (median 4.3), but was variable with an overall median of 11.1 mmHg (range 0 to 48.5). After administration of oxygen the median pO2 was 26.8 mmHg (range 0.4 to 97.6). Despite this variability in most patients there was a statistically significant (p < 0.05) response to hyperoxygenation. For example, in one patient with a breast adenocarcinoma pO2 (mmHg) measurements on days 3, 5, and 6 after implantation at baseline and with hyperoxygenation were as follows: d3 (16.4, 77.9, p< 0.01), d5 (25.5, 60.9, p <0.01), d6 (20.6, 56.8, p<0.01). Measurement of absolute pO2 via EPR oximetry in patients receiving surgical resection of their malignancies, with or without neoadjuvant therapy, is clinically feasible in a variety of tumors. Tissue oxygenation at baseline was significantly different between patients, with only a subset demonstrating clinically significant hypoxia (< 10 mm Hg). Although the majority of patients responded to hyperoxygenation interventions, some were non-responders. These data demonstrate the need for individualized assessment of tumor oxygenation in the context of planned hyperoxygenation intervention in order to optimize clinical outcomes.

Instrumentation and application methodologies for rapidly and accurately estimating individual ionizing radiation dose are needed for on-site triage in a radiological/nuclear event. One such methodology is an in vivo X-band, electron paramagnetic resonance, physically based dosimetry method to directly measure the radiation-induced signal in fingernails. The primary components under development are key instrument features, such as resonators with unique geometries that allow for large sampling volumes but limit radiation-induced signal measurements to the nail plate, and methodological approaches for addressing interfering signals in the nail and for calibrating dose from radiation-induced signal measurements. One resonator development highlighted here is a surface resonator array designed to reduce signal detection losses due to the soft tissues underlying the nail plate. Several surface resonator array geometries, along with ergonomic features to stabilize fingernail placement, have been tested in tissue-equivalent nail models and in vivo nail measurements of healthy volunteers using simulated radiation-induced signals in their fingernails. These studies demonstrated radiation-induced signal detection sensitivities and quantitation limits approaching the clinically relevant range of ≤ 10 Gy. Studies of the capabilities of the current instrument suggest that a reduction in the variability in radiation-induced signal measurements can be obtained with refinements to the surface resonator array and ergonomic features of the human interface to the instrument. Additional studies are required before the quantitative limits of the assay can be determined for triage decisions in a field application of dosimetry. These include expanded in vivo nail studies and associated ex vivo nail studies to provide informed approaches to accommodate for a potential interfering native signal in the nails when calculating the radiation-induced signal from the nail plate spectral measurements and to provide a method for calibrating dose estimates from the radiation-induced signal measurements based on quantifying experiments in patients undergoing total-body irradiation or total-skin electron therapy.

Purpose Transcutaneous oxygen tension (TcpO 2 ) provides information about blood perfusion in the tissue immediately below the skin. These data are valuable in assessing wound healing problems, diagnosing peripheral vascular/arterial insufficiency, and predicting disease progression or the response to therapy. Currently, TcpO 2 is primarily measured using electrochemical skin sensors, which consume oxygen and are prone to calibration errors. The goal of the present study was to develop a reliable method for TcpO 2 measurement in human subjects. Methods We have developed a novel TcpO 2 oximetry method based on electron paramagnetic resonance (EPR) principles with an oxygen‐sensing skin adhesive film, named the superficial perfusion oxygen tension (SPOT) chip. The SPOT chip is a 3‐mm diameter, 60‐μm thick circular film composed of a stable paramagnetic oxygen sensor. The chip is covered with an oxygen‐barrier material on one side and secured on the skin by a medical adhesive transfer tape to ensure that only the oxygen that diffuses through the skin surface is measured. The method quantifies TcpO 2 through the linewidth of the EPR spectrum. Results Repeated measurements using a cohort of 10 healthy human subjects showed that the TcpO 2 measurements were robust, reliable, and reproducible. The TcpO 2 values ranged from 7.8 ± 0.8 to 22.0 ± 1.0 mmHg in the volar forearm skin ( N = 29) and 8.1 ± 0.3 to 23.4 ± 1.3 mmHg in the foot ( N = 86). Conclusions The results demonstrated that the SPOT chip can measure TcpO 2 reliably and repeatedly under ambient conditions. The SPOT chip method could potentially be used to monitor TcpO 2 in the clinic.

Hypoxic tumors are more resistant to radiotherapy and chemotherapy, which decreases the efficacy of these common forms of treatment. We have been developing implantable paramagnetic particulates to measure oxygen in vivo using electron paramagnetic resonance. Once implanted, oxygen can be measured repeatedly and non-invasively in superficial tissues (<3 cm deep), using an electron paramagnetic resonance spectrometer and an external surface-loop resonator. To significantly extend the clinical applications of electron paramagnetic resonance oximetry, we developed an implantable resonator system to obtain measurements at deeper sites. This system has been used to successfully obtain oxygen measurements in animal studies for several years. We report here on recent developments needed to meet the regulatory requirements to make this technology available for clinical use. radio frequency heating is discussed and magnetic resonance compatibility testing of the device has been carried out by a Good Laboratory Practice-certified laboratory. The geometry of the implantable resonator has been modified to meet our focused goal of verifying safety and efficacy for the proposed use of intracranial measurements and also for future use in tissue sites other than the brain. We have encapsulated the device within a smooth cylindrical-shaped silicone elastomer to prevent tissues from adhering to the device and to limit perturbation of tissue during implantation and removal. We have modified the configuration for simultaneously measuring oxygen at multiple sites by developing a linear array of oxygen sensing probes, which each provide independent measurements. If positive results are obtained in additional studies which evaluate biocompatibility and chemical characterization, we believe the implantable resonator will be at a suitable stage for initial testing in human subjects.

A new resonator for X-band electron paramagnetic resonance (EPR) spectroscopy, which utilizes the unique resonance properties of dielectric substrates, has been developed using a single crystal of titanium dioxide. As a result of the dielectric properties of the crystal(s) chosen, this novel resonator provides the ability to make in vivo EPR spectroscopy surface measurements in the presence of lossy tissues at X-band frequencies (up to 10 GHz). A double-loop coupling device is used to transmit and receive microwave power to/from the resonator. This coupler has been developed and optimized for coupling to the resonator in the presence of lossy tissues to further enable in vivo measurements, such as in vivo EPR spectroscopy of human fingernails or teeth to measure the dose of ionizing radiation that a given individual has been exposed to. An advantage of this resonator for surface measurements is that the magnetic fields generated by the resonator are inherently shallow, which is desirable for in vivo nail dosimetry.

Managing radiation injuries following a catastrophic event where large numbers of people may have been exposed to life-threatening doses of ionizing radiation relies on the availability of biodosimetry to assess whether individuals need to be triaged for care. Electron Paramagnetic Resonance (EPR) tooth dosimetry is a viable method to accurately estimate the amount of ionizing radiation to which an individual has been exposed. In the intended measurement conditions and scenario, it is essential that the measurement process be fast, straightforward and provides meaningful and accurate dose estimations for individuals in the expected measurement conditions. The sensing component of a conventional L-band EPR spectrometer used for tooth dosimetry typically consists of a surface coil resonator that is rigidly, physically attached to the coupler. This design can result in cumbersome operation, limitations in teeth geometries that may be measured and hinder the overall utility of the dosimeter. A novel surface coil resonator has been developed for the currently existing L-band (1.15 GHz) EPR tooth dosimeter for the intended use as a point of care device by minimally trained operators. This resonator development provides further utility to the dosimeter, and increases the usability of the dosimeter by non-expert operators in the intended use scenario.

This paper describes an optimized design of a surface coil resonator for in vivo electron paramagnetic resonance (EPR)-based tooth dosimetry. Using the optimized resonator, dose estimates with the standard error of the mean of approximately 0.5 Gy were achieved with irradiated human teeth. The product of the quality factor and the filling factor of the resonator was computed as an index of relative signal intensity in EPR tooth dosimetry by the use of 3-D electromagnetic wave simulator and radio frequency circuit design environment (ANSYS HFSS and Designer). To verify the simulated results of the signal intensity in our numerical model of the resonator and a tooth sample, we experimentally measured the radiation-induced signals from an irradiated tooth with an optimally designed resonator. In addition to the optimization of the resonator design, we demonstrated the improvement of the stability of EPR spectra by decontamination of the surface coil resonator using an HCl solution, confirming that contamination of small magnetic particles on the silver wire of the surface coil had degraded the stability of the EPR spectral baseline.

A variable radio frequency proton-electron double-resonance imaging (VRF PEDRI) approach for pH mapping of aqueous samples has been recently developed (Efimova et al. J. Magn. Reson. 2011, 209, 227-232). A pH map is extracted from two PEDRI acquisitions performed at electron paramagnetic resonance (EPR) frequencies of protonated and unprotonated forms of a pH-sensitive probe. To translate VRF PEDRI to an in vivo setting, an advanced pH probe was synthesized. Probe deuteration resulted in a narrow spectral line of 1.2 G compared to a nondeuterated analogue line width of 2.1 G allowing for an increase of Overhauser enhancements and reduction in rf power deposition. Binding of the probe to the cell-impermeable tripeptide, glutathione (GSH), allows for targeting to extracellular tissue space for monitoring extracellular tumor acidosis, a prognostic factor in tumor pathophysiology. The probe demonstrated pH sensitivity in the 5.8-7.8 range, optimum for measurement of acidic extracellular tumor pH (pH(e)). In vivo VRF PEDRI was performed on Met-1 tumor-bearing mice. Compared to normal mammary glands with a neutral mean pH(e) (7.1 ± 0.1), we observed broader pH distribution with acidic mean pH(e) (6.8 ± 0.1) in tumor tissue. In summary, VRF PEDRI in combination with a newly developed pH probe provides an analytical approach for spatially resolved noninvasive pHe monitoring, in vivo.

Approach for in vivo real-time assessment of tumor tissue extracellular pH (pH(e)), redox, and intracellular glutathione based on L-band EPR spectroscopy using dual function pH and redox nitroxide probe and disulfide nitroxide biradical, is described. These parameters were monitored in PyMT mice bearing breast cancer tumors during treatment with granulocyte macrophage colony-stimulating factor. It was observed that tumor pH(e) is about 0.4 pH units lower than that in normal mammary gland tissue. Treatment with granulocyte macrophage colony-stimulating factor decreased the value of pH(e) by 0.3 units compared with PBS control treatment. Tumor tissue reducing capacity and intracellular glutathione were elevated compared with normal mammary gland tissue. Granulocyte macrophage colony-stimulating factor treatment resulted in a decrease of the tumor tissue reducing capacity and intracellular glutathione content. In addition to spectroscopic studies, pH(e) mapping was performed using recently proposed variable frequency proton-electron double-resonance imaging. The pH mapping superimposed with MRI image supports probe localization in mammary gland/tumor tissue, shows high heterogeneity of tumor tissue pH(e) and a difference of about 0.4 pH units between average pH(e) values in tumor and normal mammary gland. In summary, the developed multifunctional approach allows for in vivo, noninvasive pH(e), extracellular redox, and intracellular glutathione content monitoring during investigation of various therapeutic strategies for solid tumors.

To develop and evaluate a two‐dimensional (2D) fast spin echo (FSE) pulse sequence for enhancing temporal resolution and reducing tissue heating for in vivo proton electron double resonance imaging (PEDRI) of mice.