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We report the development of a high-sensitivity and high-resolution PET subsystem for a next-generation preclinical PET/EPR hybrid scanner for investigating and improving hypoxia imaging with PET. The PET subsystem consists of 14 detector modules (DM) installed within a cylindrical supporting frame whose outer and inner diameters are 115mm and 60mm, respectively. Each DM contains eight detector units (DU) in a row and each DU is made of a 12×12 array of 1×1×10mm 3 LYSO crystals (with a 1.05mm pitch) coupled to a 4×4 silicon photomultiplier (SiPM) array that has a 3.2mm pitch (Hamamatsu multi-pixel photon counter (MPPC) array 14161-3050HS-04). The PET subsystem has a 104mm axial field-of-view (AFOV) that is sufficient for full-body mouse imaging, therefore enabling temporal and spatial correlation studies of tumor hypoxia between PET and EPR. It employs 1mm-width crystals to support sub-millimeter image resolution that is desired for mouse imaging. Al-though a DM contains 1,152 LYSO crystals, by use of a newly devised signal readout method only six outputs are produced. Recently a partial prototype of this subsystem consisting of four DMs is built. In this paper, we present performance measurement results obtained for the developed DMs and initial imaging results obtained by the prototype. The developed DMs show uniformly superior performance in identifying the hit crystal and detector unit, in energy resolution, and in coincidence time resolution. The images obtained for a 22 Na point source and a 18 F-filled U-shaped tube source show an image resolution of about 1.1mm and 1.2mm FWHM in the transverse and axial directions respectively, and demonstrate successful imaging over the entire 104mm AFOV of the prototype. This estimated image resolution however includes the contribution by the source size., Competing Interests: Declaration of Interest Statement We have no conflicts of interest to disclose. Heejong Kim On behalf of the co-authors

Purpose: Progress toward developing a novel radiocontrast agent for determining pO 2 in tumors in a clinical setting is described. The imaging agent is designed for use with electron paramagnetic resonance imaging (EPRI), in which the collision of a paramagnetic probe molecule with molecular oxygen causes a spectroscopic change which can be calibrated to give the real oxygen concentration in the tumor tissue., Procedures: The imaging agent is based on a nanoscaffold of aluminum hydroxide (boehmite) with sizes from 100 to 200 nm, paramagnetic probe molecule, and encapsulation with a gas permeable, thin (10-20 nm) polymer layer to separate the imaging agent and body environment while still allowing O 2 to interact with the paramagnetic probe. A specially designed deuterated Finland trityl (dFT) is covalently attached on the surface of the nanoparticle through 1,3-dipolar addition of the alkyne on the dFT with an azide on the surface of the nanoscaffold. This click-chemistry reaction affords 100% efficiency of the trityl attachment as followed by the complete disappearance of the azide peak in the infrared spectrum. The fully encapsulated, dFT-functionalized nanoparticle is referred to as RADI-Sense., Results: Side-by-side in vivo imaging comparisons made in a mouse model made between RADI-Sense and free paramagnetic probe (OX-071) showed oxygen sensitivity is retained and RADI-Sense can create 3D pO 2 maps of solid tumors CONCLUSIONS: A novel encapsulated nanoparticle EPR imaging agent has been described which could be used in the future to bring EPR imaging for guidance of radiotherapy into clinical reality., (© 2023. The Author(s), under exclusive licence to World Molecular Imaging Society.)

Purpose: Spatial heterogeneity in tumor hypoxia is one of the most important factors regulating tumor growth, development, aggressiveness, metastasis, and affecting treatment outcome. Most solid tumors are known to have hypoxia or low oxygen levels (pO 2 ≤10 torr). Electron paramagnetic resonance oxygen imaging (EPROI) is an emerging oxygen mapping technology. EPROI utilizes the linear relationship between the relaxation rates of the injectable OX071 trityl spin probe and the partial oxygen pressure (pO 2 ). However, most of the EPROI studies have been limited to mouse models of solid tumors because of the instrument-size limitations. The purpose of this work was to develop a human-sized 9-mT (250 MHz resonance frequency, 60 cm bore size) pulse EPROI instrument and evaluate its performance with rabbit VX-2 tumor oxygen imaging., Methods: A New Zealand white rabbit with a 3.2-cm VX-2 tumor in the calf muscle was imaged using the human-sized EPROI instrument and a 2.25-in. ID volume coil. The animal received a ~8-min intravenous injection of OX071 (5.2 mL total volume at 72 mM concentration) and, after 75 min, an intratumoral injection (120 μL total at 5 mM OX071 concentration) and underwent EPROI. At the end of the experiments, MRI was performed using a preclinical 9.4-T MRI system to outline the tumor boundaries., Results: For the first time, a human-sized pulse EPROI instrument with a 60-cm bore size/250-MHz frequency was built and evaluated using rabbit tumor oxygen imaging. For the first time, the systemic IV injection of the oxygen-sensitive trityl OX071 spin probe was used for an animal of this size. The resulting EPROI image from the IV injection showed complete tumor coverage. The image obtained after intratumoral injection showed localized coverage in the upper lobe of the tumor, demonstrating the need for improved intratumoral injection protocol., Conclusions: This study demonstrates the performance of the world's first human-sized pulse EPROI instrument. It also demonstrates that the EPROI of larger animals can be performed using the systemic injection of a manageable amount of the spin probe. This brings EPROI one step closer to clinical applications in cancer therapies. Oxygen imaging is a platform technology, and the instrument and techniques developed here will also be useful for other clinical applications., (© 2023. The Author(s), under exclusive licence to World Molecular Imaging Society.)

Purpose: The determination of blood-brain barrier (BBB) integrity and partial pressure of oxygen (pO 2 ) in the brain is of substantial interest in several neurological applications. This study aimed to assess the feasibility of using trityl OX071-based pulse electron paramagnetic resonance imaging (pEPRI) to provide a quantitative estimate of BBB integrity and pO 2 maps in mouse brains as a function of neuroinflammatory disease progression., Methods: Five Connexin-32 (Cx32)-knockout (KO) mice were injected with lipopolysaccharide to induce neuroinflammation for imaging. Three wild-type mice were also used to optimize the imaging procedure and as control animals. An additional seven Cx32-KO mice were used to establish the BBB leakage of trityl using the colorimetric assay. All pEPRI experiments were performed using a preclinical instrument, JIVA-25 (25 mT/720 MHz), at times t = 0, 4, and 6 h following lipopolysaccharide injection. Two pEPRI imaging techniques were used: (a) single-point imaging for obtaining spatial maps to outline the brain and calculate BBB leakage using the signal amplitude, and (b) inversion-recovery electron spin echo for obtaining pO 2 maps., Results: A statistically significant change in BBB leakage was found using pEPRI with the progression of inflammation in Cx32 KO animals. However, the change in pO 2 values with the progression of inflammation for these animals was not statistically significant., Conclusions: For the first time, we show the ability of pEPRI to provide pO 2 maps in mouse brains noninvasively, along with a quantitative assessment of BBB leakage. We expect this study to open new queries from the field to explore the pathology of many neurological diseases and provide a path to new treatments., (© 2024 International Society for Magnetic Resonance in Medicine.)

Purpose: Tumor hypoxia contributes to aggressive phenotypes and diminished therapeutic responses to radiation therapy (RT) with hypoxic tissue being 3-fold less radiosensitive than normoxic tissue. A major challenge in implementing hypoxic radiosensitizers is the lack of a high-resolution imaging modality that directly quantifies tissue-oxygen. The electron paramagnetic resonance oxygen-imager (EPROI) was used to quantify tumor oxygenation in two murine tumor models: E0771 syngeneic transplant breast cancers and primary p53/MCA soft tissue sarcomas, with the latter autochthonous model better recapitulating the tumor microenvironment in human malignancies. We hypothesized that tumor hypoxia differs between these models. We also aimed to quantify the absolute change in tumor hypoxia induced by the mitochondrial inhibitor papaverine (PPV) and its effect on RT response., Procedures: Tumor oxygenation was characterized in E0771 and primary p53/MCA sarcomas via EPROI, with the former model also being quantified indirectly via diffuse reflectance spectroscopy (DRS). After confirming PPV's effect on hypoxic fraction (via EPROI), we compared the effect of 0 versus 2 mg/kg PPV prior to 20 Gy on tumor growth delay and survival., Results: Hypoxic sarcomas were more radioresistant than normoxic sarcomas (p=0.0057, 2-way ANOVA), and high baseline hypoxic fraction was a significant (p=0.0063, Cox Regression Model) hazard in survivability regardless of treatment. Pre-treatment with PPV before RT did not radiosensitize tumors in the sarcoma or E0771 model. In the sarcoma model, EPROI successfully identified baseline hypoxic tumors. DRS quantification of total hemoglobin, saturated hemoglobin, changes in mitochondrial potential and glucose uptake showed no significant difference in E0771 tumors pre- and post-PPV., Conclusion: EPROI provides 3D high-resolution pO 2 quantification; EPR is better suited than DRS to characterize tumor hypoxia. PPV did not radiosensitize E0771 tumors nor p53/MCA sarcomas, which may be related to the complex pattern of vasculature in each tumor. Additionally, understanding model-dependent tumor hypoxia will provide a much-needed foundation for future therapeutic studies with hypoxic radiosensitizers., (© 2023. The Author(s), under exclusive licence to World Molecular Imaging Society.)

Purpose: This study aimed to develop a biocompatible oximetric electron paramagnetic resonance (EPR) spin probe with reduced self-relaxation, and sensitivity to oxygen for a higher signal-to-noise ratio and longer relaxation times at high oxygen concentration, compared to the reference spin probe OX071., Procedures: SOX71 was synthesized by succinylation of the twelve alcohol groups of OX071 spin probe and characterized by EPR at X-Band (9.5 GHz) and at low field (720 MHz). The biocompatibility of SOX71 was tested in vitro and in vivo in mice. A pharmacokinetic study was performed to determine the best time frame for EPR imaging. Finally, a proof-of-concept EPR oxygen imaging was performed on a mouse model of a fibrosarcoma tumor., Results: SOX71 was synthesized in one step from OX071. SOX71 exhibits a narrow line EPR spectrum with a peak-to-peak linewidth of 66 mG, similar to OX071. SOX71 does not bind to albumin nor show cell toxicity for the concentrations tested up to 5 mM. No toxicity was observed after systemic delivery via intraperitoneal injection in mice at twice the dose required for EPR imaging. After the injection, the probe is readily absorbed into the bloodstream, with a peak blood concentration half an hour, post-injection. Then, the probe is quickly cleared by the kidney with a half-life of ~ 45 min. SOX71 shows long relaxation times under anoxic condition (T 1e  = 9.5 µs and T 2e  = 5.1 µs; [SOX71] = 1 mM in PBS at 37 °C, pO 2  = 0 mmHg, 720 MHz). Both the relaxation rates R 1e and R 2e show a decreased sensitivity to pO 2, leading to twice longer relaxation times under room air conditions (pO 2  = 159 mmHg) compared to OX071. This is ideal for oxygen imaging in samples with a wide range of pO 2 . Both the relaxation rates R 1e and R 2e show a decreased sensitivity to self-relaxation compared to OX071, with a negligible effect of the probe concentration on R 1e . SOX71 was successfully applied to image oxygen in a tumor., Conclusion: SOX71, a succinylated derivative of OX071 was synthesized, characterized, and applied for in vivo EPR tumor oxygen imaging. SOX71 is highly biocompatible, and shows decreased sensitivity to oxygen and self-relaxation. This first report suggests that SOX71 is superior to OX071 for absolute oxygen mapping under a broad range of pO 2 values., (© 2023. The Author(s), under exclusive licence to World Molecular Imaging Society.)

Objective. Electron paramagnetic resonance (EPR) imaging is an advanced in vivo oxygen imaging modality. The main drawback of EPR imaging is the long scanning time. Sparse-view projections collection is an effective fast scanning pattern. However, the commonly-used filtered back projection (FBP) algorithm is not competent to accurately reconstruct images from sparse-view projections because of the severe streak artifacts. The aim of this work is to develop an advanced algorithm for sparse reconstruction of 3D EPR imaging. Methods. The optimization based algorithms including the total variation (TV) algorithm have proven to be effective in sparse reconstruction in EPR imaging. To further improve the reconstruction accuracy, we propose the directional TV (DTV) model and derive its Chambolle-Pock solving algorithm. Results. After the algorithm correctness validation on simulation data, we explore the sparse reconstruction capability of the DTV algorithm via a simulated six-sphere phantom and two real bottle phantoms filled with OX063 trityl solution and scanned by an EPR imager with a magnetic field strength of 250 G. Conclusion. Both the simulated and real data experiments show that the DTV algorithm is superior to the existing FBP and TV-type algorithms and a deep learning based method according to visual inspection and quantitative evaluations in sparse reconstruction of EPR imaging. Significance. These insights gained in this work may be used in the development of fast EPR imaging workflow of practical significance., (© 2024 Institute of Physics and Engineering in Medicine.)

Significance: Fundamental to the application of tissue redox status to human health is the quantification and localization of tissue redox abnormalities and oxidative stress and their correlation with the severity and local extent of disease to inform therapy. The centrality of the low-molecular-weight thiol, glutathione, in physiological redox balance has long been appreciated, but direct measurement of tissue thiol status in vivo has not been possible hitherto. Recent advances in instrumentation and molecular probes suggest the feasibility of real-time redox assessment in humans. Recent Advances: Recent studies have demonstrated the feasibility of using low-frequency electron paramagnetic resonance (EPR) techniques for quantitative imaging of redox status in mammalian tissues in vivo . Rapid-scan (RS) EPR spectroscopy and imaging, new disulfide-dinitroxide spin probes, and novel analytic techniques have led to significant advances in direct, quantitative imaging of thiol redox status. Critical Issues: While novel RS EPR imaging coupled with first-generation molecular probes has demonstrated the feasibility of imaging thiol redox status in vivo , further technical advancements are desirable and ongoing. These include developing spin probes that are tailored for specific tissues with response kinetics tuned to the physiological environment. Equally critical are RS instrumentation with higher signal-to-noise ratio and minimal signal distortion, as well as optimized imaging protocols for image acquisition with sparsity adapted to image information content. Future Directions: Quantitative images of tissue glutathione promise to enable acquisition of a general image of mammalian and potentially human tissue health.

Precise radiation guided by oxygen images has demonstrated superiority over the traditional radiation methods. Electron paramagnetic resonance (EPR) imaging has proven to be the most advanced oxygen imaging modality. However, the main drawback of EPR imaging is the long scan time. For each projection, we usually need to collect the projection many times and then average them to achieve high signal-to-noise ratio (SNR). One approach to fast scan is to reduce the repeating time for each projection. While the projections would be noisy and thus the traditional commonly-use filtered backprojection (FBP) algorithm would not be capable of accurately reconstructing images. Optimization-based iterative algorithms may accurately reconstruct images from noisy projections for they may incorporate prior information into optimization models. Based on the total variation (TV) algorithms for EPR imaging, in this work, we propose a directional TV (DTV) algorithm to further improve the reconstruction accuracy. We construct the DTV constrained, data divergence minimization (DTVcDM) model, derive its Chambolle-Pock (CP) solving algorithm, validate the correctness of the whole algorithm, and perform evaluations via simulated and real data. The experimental results show that the DTV algorithm outperforms the existing TV and FBP algorithms in fast EPR imaging. Compared to the standard FBP algorithm, the proposed algorithm may achieve 10 times of acceleration., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

In continuous-wave electron paramagnetic resonance imaging (CW EPRI), data are collected generally at densely sampled views sufficient for achieving accurate reconstruction of a four dimensional spectral-spatial (4DSS) image by use of the conventional filtered-backprojection (FBP) algorithm. It is desirable to minimize the scan time by collection of data only at sparsely sampled views, referred to as sparse-view data. Interest thus remains in investigation of algorithms for accurate reconstruction of 4DSS images from sparse-view data collected for potentially enabling fast data acquisition in CW EPRI. In this study, we investigate and demonstrate optimization-based algorithms for accurate reconstruction of 4DSS images from sparse-view data. Numerical studies using simulated and real sparse-view data acquired in CW EPRI are conducted that reveal, in terms of image visualization and physical-parameter estimation, the potential of the algorithms developed for yielding accurate 4DSS images from sparse-view data in CW EPRI. The algorithms developed may be exploited for enabling sparse-view scans with minimized scan time in CW EPRI for yielding 4DSS images of quality comparable to, or better than, that of the FBP reconstruction from data collected at densely sampled views., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Xiaochuan Pan reports financial support was provided by National Institutes of Health. Boris Epel, Howard Halpern reports a relationship with O2M Technologies LLC that includes: board membership and equity or stocks. Howard Halpern has patent #8664955, 10568537, and 9392957. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Cellular therapies for type-1 diabetes can leverage cell encapsulation to dispense with immunosuppression. However, encapsulated islet cells do not survive long, particularly when implanted in poorly vascularized subcutaneous sites. Here we show that the induction of neovascularization via temporary controlled inflammation through the implantation of a nylon catheter can be used to create a subcutaneous cavity that supports the transplantation and optimal function of a geometrically matching islet-encapsulation device consisting of a twisted nylon surgical thread coated with an islet-seeded alginate hydrogel. The neovascularized cavity led to the sustained reversal of diabetes, as we show in immunocompetent syngeneic, allogeneic and xenogeneic mouse models of diabetes, owing to increased oxygenation, physiological glucose responsiveness and islet survival, as indicated by a computational model of mass transport. The cavity also allowed for the in situ replacement of impaired devices, with prompt return to normoglycemia. Controlled inflammation-induced neovascularization is a scalable approach, as we show with a minipig model, and may facilitate the clinical translation of immunosuppression-free subcutaneous islet transplantation., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

We report the design and experimental validation of a compact positron emission tomography (PET) detector module (DM) intended for building a preclinical PET and electron-paramagnetic-resonance-imaging hybrid system that supports sub-millimeter image resolution and high-sensitivity, whole-body animal imaging. The DM is eight detector units (DU) in a row. Each DU contains 12×12 lutetium-yttrium oxyorthosilicate (LYSO) crystals having a 1.05 mm pitch read by 4×4 silicon photomultipliers (SiPM) having a 3.2 mm pitch. A small-footprint, highly-multiplexing readout employing only passive electronics is devised to produce six outputs for the DM, including two outputs derived from SiPM cathodes for determining event time and active DU and four outputs derived from SiPM anodes for determining energy and active crystal. Presently, we have developed two DMs that are 1.28×10.24 cm 2 in extent and approximately 1.8 cm in thickness, with their outputs sampled at 0.7 GS/s and analyzed offline. For both DMs, our results show successfully discriminated DUs and crystals. With no correction for SiPM nonlinearity, the average energy resolution for crystals in a DU ranges from 14% to 16%. While not needed for preclinical imaging, the DM may support 300-400 ps time-of-flight resolution.

Background: Clinical attempts to find benefit from specifically targeting and boosting resistant hypoxic tumor subvolumes have been promising but inconclusive. While a first preclinical murine tumor type showed significant improved control with hypoxic tumor boosts, a more thorough investigation of efficacy from boosting hypoxic subvolumes defined by electron paramagnetic resonance oxygen imaging (EPROI) is necessary. The present study confirms improved hypoxic tumor control results in three different tumor types using a clonogenic assay and explores potential confounding experimental conditions., Materials and Methods: Three murine tumor models were used for multi-modal imaging and radiotherapy: MCa-4 mammary adenocarcinomas, SCC7 squamous cell carcinomas, and FSa fibrosarcomas. Registered T2-weighted MRI tumor boundaries, hypoxia defined by EPROI as pO 2 ≤ 10 mmHg, and X-RAD 225Cx CT boost boundaries were obtained for all animals. 13 Gy boosts were directed to hypoxic or equal-integral-volume oxygenated tumor regions and monitored for regrowth. Kaplan-Meier survival analysis was used to assess local tumor control probability (LTCP). The Cox proportional hazards model was used to assess the hazard ratio of tumor progression of Hypoxic Boost vs. Oxygenated Boost for each tumor type controlling for experimental confounding variables such as EPROI radiofrequency, tumor volume, hypoxic fraction, and delay between imaging and radiation treatment., Results: An overall significant increase in LTCP from Hypoxia Boost vs. Oxygenated Boost treatments was observed in the full group of three tumor types ( p  < 0.0001). The effects of tumor volume and hypoxic fraction on LTCP were dependent on tumor type. The delay between imaging and boost treatments did not have a significant effect on LTCP for all tumor types., Conclusion: This study confirms that EPROI locates resistant tumor hypoxic regions for radiation boost, increasing clonogenic LTCP, with potential enhanced therapeutic index in three tumor types. Preclinical absolute EPROI may provide correction for clinical hypoxia images using additional clinical physiologic MRI., Competing Interests: HH holds US patent 8,664,955 (US-8664955-B1) and HH and BE hold US patent 9,392,957 (US-9392957-B1) for aspects of the pO2 imaging technology. MK, HH, and BE are members of a start-up company O2M Technologies, LLC. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Gertsenshteyn, Epel, Giurcanu, Barth, Lukens, Hall, Martinez, Grana, Maggio, Miller, Sundramoorthy, Krzykawska-Serda, Pearson, Aydogan, Weichselbaum, Tormyshev, Kotecha and Halpern.)

Type 1 diabetes (T1D) is an autoimmune disease that leads to the loss of insulin-producing beta cells. Bioartificial pancreas (BAP) or beta cell replacement strategies have shown promise in curing T1D and providing long-term insulin independence. Hypoxia (low oxygen concentration) that may occur in the BAP devices due to cell oxygen consumption at the early stages after implantation damages the cells, in addition to imposing limitations to device dimensions when translating promising results from rodents to humans. Finding ways to provide cells with sufficient oxygenation remains the major challenge in realizing BAP devices' full potential. Therefore, in vitro oxygen imaging assessment of BAP devices is crucial for predicting the devices' in vivo efficiency. Electron paramagnetic resonance oxygen imaging (EPROI, also known as electron MRI or eMRI) is a unique imaging technique that delivers absolute partial pressure of oxygen (pO 2 ) maps and has been used for cancer hypoxia research for decades. However, its applicability for assessing BAP devices has not been explored. EPROI utilizes low magnetic fields in the mT range, static gradients, and the linear relationship between the spin-lattice relaxation rate (R 1 ) of oxygen-sensitive spin probes such as trityl OX071 and pO 2 to generate oxygen maps in tissues. With the support of the Juvenile Diabetes Research Foundation (JDRF), an academic-industry partnership consortium, the "Oxygen Measurement Core" was established at O2M to perform oxygen imaging assessment of BAP devices originated from core members' laboratories. This article aims to establish the protocols and demonstrate a few examples of in vitro oxygen imaging of BAP devices using EPROI. All pO 2 measurements were performed using a recently introduced 720 MHz/25 mT preclinical oxygen imager instrument, JIVA-25™. We began by performing pO 2 calibration of the biomaterials used in BAPs at 25 mT magnetic field since no such data exist. We compared the EPROI pO 2 measurement with a single-point probe for a few selected materials. We also performed trityl OX071 toxicity studies with fibroblasts, as well as insulin-producing cells (beta TC6, MIN6, and human islet cells). Finally, we performed proof-of-concept in vitro pO 2 imaging of five BAP devices that varied in size, shape, and biomaterials. We demonstrated that EPROI is compatible with commonly used biomaterials and that trityl OX071 is nontoxic to cells. A comparison of the EPROI with a fluorescent-based point oxygen probe in selected biomaterials showed higher accuracy of EPROI. The imaging of typically heterogenous BAP devices demonstrated the utility of obtaining oxygen maps over single-point measurements. In summary, we present EPROI as a quality control tool for developing efficient cell transplantation devices and artificial tissue grafts. Although the focus of this work is encapsulation systems for diabetes, the techniques developed in this project are easily transferable to other biomaterials, tissue grafts, and cell therapy devices used in the field of tissue engineering and regenerative medicine (TERM). In summary, EPROI is a unique noninvasive tool to experimentally study oxygen distribution in cell transplantation devices and artificial tissues, which can revolutionize the treatment of degenerative diseases like T1D., (© 2023. Springer Nature Limited.)

Recent advances in our understanding of hypoxia and hypoxia-mediated mechanisms shed light on the critical implications of the hypoxic stress on cellular behavior. However, tools emulating hypoxic conditions ( i.e. , low oxygen tensions) for research are limited and often suffer from major shortcomings, such as lack of reliability and off-target effects, and they usually fail to recapitulate the complexity of the tissue microenvironment. Fortunately, the field of biomaterials is constantly evolving and has a central role to play in the development of new technologies for conducting hypoxia-related research in several aspects of biomedical research, including tissue engineering, cancer modeling, and modern drug screening. In this perspective, we provide an overview of several strategies that have been investigated in the design and implementation of biomaterials for simulating or inducing hypoxic conditions-a prerequisite in the stabilization of hypoxia-inducible factor (HIF), a master regulator of the cellular responses to low oxygen. To this end, we discuss various advanced biomaterials, from those that integrate hypoxia-mimetic agents to artificially induce hypoxia-like responses, to those that deplete oxygen and consequently create either transient (<1 day) or sustained (>1 day) hypoxic conditions. We also aim to highlight the advantages and limitations of these emerging biomaterials for biomedical applications, with an emphasis on cancer research., Competing Interests: B. E. and M. K. disclose financial interests in O2M Technologies. All other authors have no conflicts of interest to disclose., (This journal is © The Royal Society of Chemistry.)

Objective: We investigate and develop optimization-based algorithms for accurate reconstruction of four-dimensional (4D)-spectral-spatial (SS) images directly from data collected over limited angular ranges (LARs) in continuous-wave (CW) electron paramagnetic resonance imaging (EPRI)., Methods: Basing on a discrete-to-discrete data model devised in CW EPRI employing the Zeeman-modulation (ZM) scheme for data acquisition, we first formulate the image reconstruction problem as a convex, constrained optimization program that includes a data fidelity term and also constraints on the individual directional total variations (DTVs) of the 4D-SS image. Subsequently, we develop a primal-dual-based DTV algorithm, simply referred to as the DTV algorithm, to solve the constrained optimization program for achieving image reconstruction from data collected in LAR scans in CW-ZM EPRI., Results: We evaluate the DTV algorithm in simulated- and real-data studies for a variety of LAR scans of interest in CW-ZM EPRI, and visual and quantitative results of the studies reveal that 4D-SS images can be reconstructed directly from LAR data, which are visually and quantitatively comparable to those obtained from data acquired in the standard, full-angular-range (FAR) scan in CW-ZM EPRI., Conclusion: An optimization-based DTV algorithm is developed for accurately reconstructing 4D-SS images directly from LAR data in CW-ZM EPRI. Future work includes the development and application of the optimization-based DTV algorithm for reconstructions of 4D-SS images from FAR and LAR data acquired in CW EPRI employing schemes other than the ZM scheme., Significance: The DTV algorithm developed may be exploited potentially for enabling and optimizing CW EPRI with minimized imaging time and artifacts by acquiring data in LAR scans., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [Authors HH and BE declare US patents 8,664,955, 10,568,537, and 9,392,957 on aspects of the pO2 imaging technology and membership in a start up company O2M Technologies. Other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.], (Copyright © 2023 Elsevier Inc. All rights reserved.)

Electron paramagnetic resonance (EPR) imaging is an advanced oxygen imaging modality for oxygen-image guided radiation. The iterative reconstruction algorithm is the research hot-point in image reconstruction for EPR imaging (EPRI) for this type of algorithm may incorporate image-prior information to construct advanced optimization model to achieve accurate reconstruction from sparse-view projections and/or noisy projections. However, the system matrix in the iterative algorithm needs complicated calculation and needs huge memory-space if it is stored in memory. In this work, we propose an iterative reconstruction algorithm without system matrix for EPRI to simplify the whole iterative reconstruction process. The function of the system matrix is to calculate the projections, whereas the function of the transpose of the system matrix is to perform backprojection. The existing projection and backprojection methods are all based on the configuration that the imaged-object remains stationary and the scanning device rotates. Here, we implement the projection and backprojection operations by fixing the scanning device and rotating the object. Thus, the core algorithm is only the commonly-used image-rotation algorithm, while the calculation and store of the system matrix are avoided. Based on the idea of image rotation, we design a specific iterative reconstruction algorithm for EPRI, total variation constrained data divergence minimization (TVcDM) algorithm without system matrix, and named it as image-rotation based TVcDM (R-TVcDM). Through a series of comparisons with the original TVcDM via real projection data, we find that the proposed algorithm may achieve similar reconstruction accuracy with the original one. But it avoids the complicated calculation and store of the system matrix. The insights gained in this work may be also applied to other imaging modalities, for example computed tomography and positron emission tomography., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Purpose: To identify the optimal threshold in 18 F-fluoromisonidazole (FMISO) PET images to accurately locate tumor hypoxia by using electron paramagnetic resonance imaging (pO 2 EPRI) as ground truth for hypoxia, defined by pO 2 [Formula: see text] 10 mmHg., Methods: Tumor hypoxia images in mouse models of SCCVII squamous cell carcinoma (n = 16) were acquired in a hybrid PET/EPRI imaging system 2 h post-injection of FMISO. T2-weighted MRI was used to delineate tumor and muscle tissue. Dynamic contrast enhanced (DCE) MRI parametric images of K trans and v e were generated to model tumor vascular properties. Images from PET/EPR/MRI were co-registered and resampled to isotropic 0.5 mm voxel resolution for analysis. PET images were converted to standardized uptake value (SUV) and tumor-to-muscle ratio (TMR) units. FMISO uptake thresholds were evaluated using receiver operating characteristic (ROC) curve analysis to find the optimal FMISO threshold and unit with maximum overall hypoxia similarity (OHS) with pO 2 EPRI, where OHS = 1 shows perfect overlap and OHS = 0 shows no overlap. The means of dice similarity coefficient, normalized Hausdorff distance, and accuracy were used to define the OHS. Monotonic relationships between EPRI/PET/DCE-MRI were evaluated with the Spearman correlation coefficient ([Formula: see text]) to quantify association of vasculature on hypoxia imaged with both FMISO PET and pO 2 EPRI., Results: FMISO PET thresholds to define hypoxia with maximum OHS (both OHS = 0.728 [Formula: see text] 0.2) were SUV [Formula: see text] 1.4 [Formula: see text] SUV mean and SUV [Formula: see text] 0.6 [Formula: see text] SUV max . Weak-to-moderate correlations (|[Formula: see text]|< 0.70) were observed between PET/EPRI hypoxia images with vascular permeability (K trans ) or fractional extracellular-extravascular space (v e ) from DCE-MRI., Conclusion: This is the first in vivo comparison of FMISO uptake with pO 2 EPRI to identify the optimal FMISO threshold to define tumor hypoxia, which may successfully direct hypoxic tumor boosts in patients, thereby enhancing tumor control., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

One of the major drawbacks associated with autologous fat grafting is unpredictable graft retention. Various efforts to improve the survivability of these cells have been explored, but these methods are time-consuming, complex, and demand significant technical skill. In our study, we examine the use of cryopreserved amniotic membrane as a source of exogenous growth factors to improve adipocyte survivability under normal and hypoxic conditions. Human primary preadipocytes were cultured in a gelatin-ferulic acid (Gtn-FA) hydrogel with variable oxygen concentration and treated with amniotic membrane-derived condition medium (CM) for 7 days. This hydrogel provides a hypoxic environment and also creates a 3D cell culture to better mimic recipient site conditions. The O 2 concentration in the hydrogel was measured by electron paramagnetic resonance oxygen imaging (EPROI). The conjugation of FA was confirmed by FTIR and NMR spectroscopy. The cell viability and adipocyte differentiation were analyzed by alamarBlue™ assay, Oil Red O staining, and RT-qPCR. The expression of genes: Pref-1, C/EBP β, C/EBP α, PPAR-ƴ, SLC2A4, and VEGF-A were quantified. The cell viability results show that the 50% CM showed significantly higher cell pre-adipocyte cell viability. In addition, compared to normal conditions, hypoxia/CM provided higher PPAR-ƴ (p < .05), SLC2A4, and VEGF-A (p < .05) (early and terminal differentiating markers) mRNA expression. This finding demonstrates the efficacy of amniotic CM supplementation as a novel way to promote adipocyte survival and retention via the expression of key gene markers for differentiation and angiogenesis., (© 2022 The Authors. Journal of Biomedical Materials Research Part B: Applied Biomaterials published by Wiley Periodicals LLC.)

The purpose of this study was to assess the natural partial oxygen pressure (pO 2 ) of subcutaneous (SC) and intraperitoneal (IP) sites in mice to determine their relative suitability as sites for placement of implants. The pO 2 measurements were performed using oxygen imaging of solid probes using lithium phthalocyanine (LiPc) as the oxygen sensitive material. LiPc is a water-insoluble crystalline probe whose spin-lattice and spin-spin relaxation rates ( R 1 and R 2 ) are sensitive to the local oxygen concentration. To facilitate direct in vivo oxygen imaging, we prepared a solid probe containing encapsulated LiPc crystals in polydimethylsiloxane (PDMS), an oxygen-permeable and bioinert polymer. Although LiPc-PDMS or similar probes have been used in repeated spectroscopic or average oxygen measurements using continuous wave electron paramagnetic resonance (EPR) since the late 1990s and now have advanced to clinical applications, they have not been used for pulse EPR oxygen imaging. One LiPc-PDMS probe of 2 mm diameter and 10 mm length was implanted in SC or IP sites (left or right side) in each animal. The pO 2 imaging of implanted LiPc-PDMS probes was performed weekly for 6 weeks using O2M preclinical 25 mT oxygen imager, JIVA-25™, using the pulse inversion recovery electron spin echo method. At week 6, the probes were recovered, and histological examinations were performed. We report in this study, first-ever solid probe oxygen imaging of implanted devices and pO 2 assessment of SC and IP sites.

Inadequate oxygenation is a major challenge in cell encapsulation, a therapy which holds potential to treat many diseases including type I diabetes. In such systems, cellular oxygen (O 2 ) delivery is limited to slow passive diffusion from transplantation sites through the poorly O 2 -soluble encapsulating matrix, usually a hydrogel. This constrains the maximum permitted distance between the encapsulated cells and host site to within a few hundred micrometers to ensure cellular function. Inspired by the natural gas-phase tracheal O 2 delivery system of insects, we present herein the design of a biomimetic scaffold featuring internal continuous air channels endowed with 10,000-fold higher O 2 diffusivity than hydrogels. We incorporate the scaffold into a bulk hydrogel containing cells, which facilitates rapid O 2 transport through the whole system to cells several millimeters away from the device-host boundary. A computational model, validated by in vitro analysis, predicts that cells and islets maintain high viability even in a thick (6.6 mm) device. Finally, the therapeutic potential of the device is demonstrated through the correction of diabetes in immunocompetent mice using rat islets for over 6 months., (© 2021. The Author(s).)

Total variation (TV) minimization algorithm is an effective algorithm capable of accurately reconstructing images from sparse projection data in a variety of imaging modalities including computed tomography (CT) and electron paramagnetic resonance imaging (EPRI). The data divergence constrained, TV minimization (DDcTV) model and its Chambolle-Pock (CP) solving algorithm have been proposed for CT. However, when the DDcTV-CP algorithm is applied to 3D EPRI, it suffers from slow convergence rate or divergence. We hypothesize that this is due to the magnitude imbalance between the data fidelity term and the TV regularization term. In this work, we propose a balanced TV (bTV) model incorporating a balance parameter and demonstrate its capability to avoid convergence issues for the 3D EPRI application. Simulation and real experiments show that the DDcTV-CP algorithm cannot guarantee convergence but the bTV-CP algorithm may guarantee convergence and achieve fast convergence by use of an appropriate balance parameter. Experiments also show that underweighting the balance parameter leads to slow convergence, whereas overweighting the balance parameter leads to divergence. The iteration-behavior change-law with the variation of the balance parameter is explained by use of the data tolerance ellipse and gradient descent principle. The findings and insights gained in this work may be applied to other imaging modalities and other constrained optimization problems., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Purpose: Preclinical radiation replicating clinical intensity modulated radiation therapy (IMRT) techniques can provide data translatable to clinical practice. For this work, treatment plans were created for oxygen-guided dose-painting in small animals using inverse-planned IMRT. Spatially varying beam intensities were achieved using 3-dimensional (3D)-printed compensators., Methods and Materials: Optimized beam fluence from arbitrary gantry angles was determined using a verified model of the XRAD225Cx treatment beam. Compensators were 3D-printed with varied thickness to provide desired attenuation using copper/polylactic-acid. Spatial resolution capabilities were investigated using printed test-patterns. Following American Association of Physicists in Medicine TG119, a 5-beam IMRT plan was created for a miniaturized (∼1/8th scale) C-shape target. Electron paramagnetic resonance imaging of murine tumor oxygenation guided simultaneous integrated boost (SIB) plans conformally treating tumor to a base dose (Rx 1 ) with boost (Rx 2 ) based on tumor oxygenation. The 3D-printed compensator intensity modulation accuracy and precision was evaluated by individually delivering each field to a phantom containing radiochromic film and subsequent per-field gamma analysis. The methodology was validated end-to-end with composite delivery (incorporating 3D-printed tungsten/polylactic-acid beam trimmers to reduce out-of-field leakage) of the oxygen-guided SIB plan to a phantom containing film and subsequent gamma analysis., Results: Resolution test-patterns demonstrate practical printer resolution of ∼0.7 mm, corresponding to 1.0 mm bixels at the isocenter. The miniaturized C-shape plan provides planning target volume coverage (V 95% = 95%) with organ sparing (organs at risk D max < 50%). The SIB plan to hypoxic tumor demonstrates the utility of this approach (hypoxic tumor V 95%,Rx2 = 91.6%, normoxic tumor V 95%,Rx1 = 95.7%, normal tissue V 100%,Rx1 = 7.1%). The more challenging SIB plan to boost the normoxic tumor rim achieved normoxic tumor V 95%,Rx2 = 90.9%, hypoxic tumor V 95%,Rx1 = 62.7%, and normal tissue V 100%,Rx2 = 5.3%. Average per-field gamma passing rates using 3%/1.0 mm, 3%/0.7 mm, and 3%/0.5 mm criteria were 98.8% ± 2.8%, 96.6% ± 4.1%, and 90.6% ± 5.9%, respectively. Composite delivery of the hypoxia boost plan and gamma analysis (3%/1 mm) gave passing results of 95.3% and 98.1% for the 2 measured orthogonal dose planes., Conclusions: This simple and cost-effective approach using 3D-printed compensators for small-animal IMRT provides a methodology enabling preclinical studies that can be readily translated into the clinic. The presented oxygen-guided dose-painting demonstrates that this methodology will facilitate studies driving much needed biologic personalization of radiation therapy for improvements in patient outcomes., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Cell encapsulation represents a promising therapeutic strategy for many hormone-deficient diseases such as type 1 diabetes (T1D). However, adequate oxygenation of the encapsulated cells remains a challenge, especially in the poorly oxygenated subcutaneous site. Here, we present an encapsulation system that generates oxygen (O 2 ) for the cells from their own waste product, carbon dioxide (CO 2 ), in a self-regulated (i.e., "inverse breathing") way. We leveraged a gas-solid (CO 2 -lithium peroxide) reaction that was completely separated from the aqueous cellular environment by a gas permeable membrane. O 2 measurements and imaging validated CO 2 -responsive O 2 release, which improved cell survival in hypoxic conditions. Simulation-guided optimization yielded a device that restored normoglycemia of immunocompetent diabetic mice for over 3 months. Furthermore, functional islets were observed in scaled-up device implants in minipigs retrieved after 2 months. This inverse breathing device provides a potential system to support long-term cell function in the clinically attractive subcutaneous site., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Purpose: To enhance the spatial accuracy of fluorine 18 ( 18 F) misonidazole (MISO) PET imaging of hypoxia by using dynamic contrast-enhanced (DCE) MR images as a basis for modifying PET images and by using electron paramagnetic resonance (EPR) partial oxygen pressure (pO 2 ) as the reference standard., Materials and Methods: Mice ( n = 10) with leg-borne MCa4 mammary carcinomas underwent EPR imaging, T2-weighted and DCE MRI, and 18 F-MISO PET/CT. Images were registered to the same space for analysis. The thresholds of hypoxia for PET and EPR images were tumor-to-muscle ratios greater than or equal to 2.2 mm Hg and less than or equal to 14 mm Hg, respectively. The Dice similarity coefficient (DSC) and Hausdorff distance (d H ) were used to quantify the three-dimensional overlap of hypoxia between pO 2 EPR and 18 F-MISO PET images. A training subset ( n = 6) was used to calculate optimal DCE MRI weighting coefficients to relate EPR to the PET signal; the group average weights were then applied to all tumors (from six training mice and four test mice). The DSC and d H were calculated before and after DCE MRI-corrected PET images were obtained to quantify the improvement in overlap with EPR pO 2 images for measuring tumor hypoxia., Results: The means and standard deviations of the DSC and d H between hypoxic regions in original PET and EPR images were 0.35 mm ± 0.23 and 5.70 mm ± 1.7, respectively, for images of all 10 mice. After implementing a preliminary DCE MRI correction to PET data, the DSC increased to 0.86 mm ± 0.18 and the d H decreased to 2.29 mm ± 0.70, showing significant improvement ( P < .001) for images of all 10 mice. Specifically, for images of the four independent test mice, the DSC improved with correction from 0.19 ± 0.28 to 0.80 ± 0.29 ( P = .02), and the d H improved from 6.40 mm ± 2.5 to 1.95 mm ± 0.63 ( P = .01)., Conclusion: Using EPR information as a reference standard, DCE MRI information can be used to correct 18 F-MISO PET information to more accurately reflect areas of hypoxia. Keywords: Animal Studies, Molecular Imaging, Molecular Imaging-Cancer, PET/CT, MR-Dynamic Contrast Enhanced, MR-Imaging, PET/MR, Breast, Oncology, Tumor Mircoenvironment, Electron Paramagnetic Resonance Supplemental material is available for this article. © RSNA, 2021., Competing Interests: Disclosures of Conflicts of Interest: I.G. disclosed no relevant relationships. B.E. Activities related to the present article: disclosed no relevant relationships. Activities not related to the present article: disclosed no relevant relationships. Other relationships: co-owner of O2M Technologies, a company that fabricates EPR imaging instruments. No O2M instruments, IP, or technology was used in the present work. E.B. disclosed no relevant relationships. L.L. disclosed no relevant relationships. E.M. disclosed no relevant relationships. H.M.T. disclosed no relevant relationships. X.F. disclosed no relevant relationships. M.G. disclosed no relevant relationships. D.B. Activities related to the present article: National Cancer Institute grant R01CA236385 was used to partially fund the author’s salary for this project. Activities not related to the present article: money from O2M Technologies paid was part of the author’s transition to employment with O2M. These funds were not a part of the relevant work. Other relationships: disclosed no relevant relationships. M.Z. disclosed no relevant relationships. S.S. disclosed no relevant relationships. H.K. disclosed no relevant relationships. R.F. disclosed no relevant relationships. M.B. disclosed no relevant relationships. A.K. disclosed no relevant relationships. G.K. disclosed no relevant relationships. C.M.K. disclosed no relevant relationships. H.H. Activities related to the present article: disclosed no relevant relationships. Activities not related to the present article: disclosed no relevant relationships. Other relationships: Inventor of several patents, including Magnetic Resonance Analysis of Substances in Samples that Include Dissipative Material (inventor: Howard Halpern; patent no.: 4,714,886); Selective Isotopic Labelling of Spin Labels for Electron Resonance Spectroscopy (inventors: Howard Halpern, Beverly A. Teicher; patent no.: 5,431,901) Imaging system performing substantially exact reconstruction and using non-traditional trajectories (inventors: Pan X, Zou, Yu, Yu L, Kao; C-M, King M, Giger M, Xia D, Halpern H, Pelizzari C, Sidky E, Cho S; patent no: 7,444,011); High isolation transmit/receive surface coils and method for EPRI (inventor: Halpern H; patent no. 8,644,955); T1-Senstivie inversion recovery-imaging method and apparatus for EPRI (inventors: Halpern H, Epel B; patent no.: 9,392,957); Method and apparatus for resonator signal production and measurement (inventors: Halpern H, Sundramoorthy S, Epel B; patent no.:10,551,450B2); Method and high isolation transmit/receive surface coils for EPRI (inventor: Howard J. Halpern; patent no.: 10,568,537); and Alderman-Grant/Loop-Gap High Isolation Crossed Field Bimodal Resonator (inventors: Halpern H, Epel B, Sundramoorthy S; submitted: 4/12/2011). C.T.C. Activities related to the present article: disclosed no relevant relationships. Activities not related to the present article: royalties and stock options from RefleXion Medical, which is licensed from the University of Chicago for the author’s patent on PET or SPECT-guided radiation therapy. Other relationships: disclosed no relevant relationships., (2021 by the Radiological Society of North America, Inc.)

Yakov Sergeevich Lebedev was a pioneer in high frequency EPR, taking advantage of the separation of g-factor anisotropy effects from nuclear hyperfine splitting and the higher frequency molecular motion sensitivity from higher frequency measurements 8 . This article celebrates a second EPR subfield in which Prof. Lebedev pioneered, EPR imaging. 9 We celebrate the clinical enhancements that are suggested in this low frequency work and imaging application to animal physiology at lower-than-standard EPR frequencies.

This paper presents a survey of electron paramagnetic resonance (EPR) image registration. Image registration is the process of overlaying images (two or more) of the same scene taken at different times, from different viewpoints and/or different techniques. EPR-imaging (EPRI) techniques belong to the functional-imaging modalities and therefore suffer from a lack of anatomical reference which is mandatory in preclinical imaging. For this reason, it is necessary to merging EPR images with other modalities which allow for obtaining anatomy images. Methodological analysis and review of the literature were done, providing a summary for developing a good foundation for research study in this field which is crucial in understanding the existing levels of knowledge. Out of these considerations, the aim of this paper is to enhance the scientific community's understanding of the current status of research in EPR preclinical image registration and also communicate to them the contribution of this research in the field of image processing.

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Purpose: Tumor oxygenation is a critical parameter influencing the efficacy of cancer therapy. Low levels of oxygen in solid tumor have been recognized as an indicator of malignant progression and metastasis, as well as poor response to chemo- and radiation therapy. Being able to measure oxygenation for an individual's tumor would provide doctors with a valuable way of identifying optimal treatments for patients. Methods: Electron paramagnetic resonance imaging (EPRI) in combination with an oxygen-measuring paramagnetic probe was performed to measure tumor oxygenation in vivo. Triarylmethyl (trityl) radical exhibits high specificity, sensitivity, and resolution for quantitative measurement of O 2 concentration. However, its in vivo applications in previous studies have been limited by the required high dosage, its short half-life, and poor intracellular permeability. To address these limitations, we developed high-capacity nanoformulated radicals that employed fluorescein isothiocyanate-labeled mesoporous silica nanoparticles (FMSNs) as trityl radical carriers. The high surface area nanostructure and easy surface modification of physiochemical properties of FMSNs enable efficient targeted delivery of highly concentrated, nonself-quenched trityl radicals, protected from environmental degradation and dilution. Results: We successfully designed and synthesized a tumor-targeted nanoplatform as a carrier for trityl. In addition, the nanoformulated trityl does not affect oxygen-sensing capacity by a self-relaxation or broadening effect. The FMSN-trityl exhibited high sensitivity/response to oxygen in the partial oxygen pressure range from 0 to 155 mmHg. Furthermore, MSN-trityl displayed outstanding intracellular oxygen mapping in both in vitro and in vivo animal studies. Conclusion: The highly sensitive nanoformulated trityl spin probe can profile intracellular oxygen distributions of tumor in a real-time and quantitative manner using in vivo EPRI., Competing Interests: Prof. Dr. Leu-Wei Lo reports grants from National Health Research Institutes, Taiwan and the Ministry of Science and Technology of Taiwan. Dr. Nai-Tzu Chen reports grants from China Medical University, Taiwan, Chicago Biomedical Consortium, and the Ministry of Science and Technology of Taiwan. Prof. Dr. Howard J Halpern reports grants from USA NIH, during the conduct of the study and has patents 8,664,955, 9,392,957, 4,714,886, and 5,431,901 issued. The other authors report no conflicts of interest in this work.

Purpose: It has been known for over 100 years that tumor hypoxia, a near-universal characteristic of solid tumors, decreases the curative effectiveness of radiation therapy. However, to date, there are no reports that demonstrate an improvement in radiation effectiveness in a mammalian tumor on the basis of tumor hypoxia localization and local hypoxia treatment., Methods and Materials: For radiation targeting of hypoxic subregions in mouse fibrosarcoma, we used oxygen images obtained using pulse electron paramagnetic resonance pO 2 imaging combined with 3D-printed radiation blocks. This achieved conformal radiation delivery to all hypoxic areas in FSa fibrosarcomas in mice., Results: We demonstrate that treatment delivering a radiation boost to hypoxic volumes has a significant (P = .04) doubling of tumor control relative to boosts to well-oxygenated volumes. Additional dose to well-oxygenated tumor regions minimally increases tumor control beyond the 15% control dose to the entire tumor. If we can identify portions of the tumor that are more resistant to radiation, it might be possible to reduce the dose to more sensitive tumor volumes without significant compromise in tumor control., Conclusions: This work demonstrates in a single, intact mammalian tumor type that tumor hypoxia is a local tumor phenomenon whose treatment can be enhanced by local radiation. Despite enormous clinical effort to overcome hypoxic radiation resistance, to our knowledge this is the first such demonstration, even in preclinical models, of targeting additional radiation to hypoxic tumor to improve the therapeutic ratio., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Tumor cure with conventional fractionated radiotherapy is 65%, dependent on tumor cell-autonomous gradual buildup of DNA double-strand break (DSB) misrepair. Here we report that single-dose radiotherapy (SDRT), a disruptive technique that ablates more than 90% of human cancers, operates a distinct dual-target mechanism, linking acid sphingomyelinase-mediated (ASMase-mediated) microvascular perfusion defects to DNA unrepair in tumor cells to confer tumor cell lethality. ASMase-mediated microcirculatory vasoconstriction after SDRT conferred an ischemic stress response within parenchymal tumor cells, with ROS triggering the evolutionarily conserved SUMO stress response, specifically depleting chromatin-associated free SUMO3. Whereas SUMO3, but not SUMO2, was indispensable for homology-directed repair (HDR) of DSBs, HDR loss of function after SDRT yielded DSB unrepair, chromosomal aberrations, and tumor clonogen demise. Vasoconstriction blockade with the endothelin-1 inhibitor BQ-123, or ROS scavenging after SDRT using peroxiredoxin-6 overexpression or the SOD mimetic tempol, prevented chromatin SUMO3 depletion, HDR loss of function, and SDRT tumor ablation. We also provide evidence of mouse-to-human translation of this biology in a randomized clinical trial, showing that 24 Gy SDRT, but not 3×9 Gy fractionation, coupled early tumor ischemia/reperfusion to human cancer ablation. The SDRT biology provides opportunities for mechanism-based selective tumor radiosensitization via accessing of SDRT/ASMase signaling, as current studies indicate that this pathway is tractable to pharmacologic intervention.

Electron paramagnetic resonance imaging (EPRI) can yield information about the 3-dimensional (3D) spatial distribution of the unpaired-electron-spin density from which the spatial distribution of oxygen concentration within tumor tissue, referred to as the oxygen image or electron paramagnetic resonance (EPR) image in this work, can be derived. Existing algorithms for reconstruction of EPR images often require data collected at a large number of densely sampled projection views, resulting in a prolonged data-acquisition time and consequently numerous practical challenges especially to in vivo animal EPRI. Therefore, a strong interest exists in shortening data-acquisition time through reducing the number of data samples collected in EPRI, and one approach is to acquire data at a reduced number of sparsely distributed projection views from which existing algorithms may reconstruct images with prominent artifacts. In this work, we investigate and develop an optimization-based technique for image reconstruction from data collected at sparsely sampled projection views for reducing scanning time in EPRI. Specifically, we design a convex optimization program in which the EPR image of interest is formulated as a solution and then tailor the Chambolle-Pock (CP) primal-dual algorithm to reconstruct the image by solving the convex optimization program. Using computer-simulated EPRI data from numerical phantoms and real EPRI data collected from physical phantoms, we perform studies on the verification and characterization of the optimization-based technique for EPR image reconstruction. Results of the studies suggest that the technique may yield accurate EPR images from data collected at sparsely distributed projection views, thus potentially enabling fast EPRI with reduced acquisition time., (Copyright © 2018 Elsevier Inc. All rights reserved.)

We present a 34 GHz continuous wave (CW)/pulsed electron paramagnetic resonance (EPR) spectrometer capable of pulse-shaping that is based on a versatile microwave bridge design. The bridge radio frequency (RF)-in/RF-out design (500 MHz to 1 GHz input/output passband, 500 MHz instantaneous input/output bandwidth) creates a flexible platform with which to compare a variety of excitation and detection methods utilizing commercially available equipment external to the bridge. We use three sources of RF input to implement typical functions associated with CW and pulse EPR spectroscopic measurements. The bridge output is processed via high speed digitizer and an in-phase/quadrature (I/Q) demodulator for pulsed work or sent to a wideband, high dynamic range log detector for CW. Combining this bridge with additional commercial hardware and new acquisition and control electronics, we have designed and constructed an adaptable EPR spectrometer that builds upon previous work in the literature and is functionally comparable to other available systems., (Published by Elsevier Inc.)

Objectives: This work aims to explore more accurate pixel-driven projection methods for iterative image reconstructions in order to reduce high-frequency artifacts in the generated projection image., Methods: Three new pixel-driven projection methods namely, small-pixel-large-detector (SPLD), linear interpolation based (LIB) and distance anterpolation based (DAB), were proposed and applied to reconstruct images. The performance of these methods was evaluated in both two-dimensional (2D) computed tomography (CT) images via the modified FORBILD phantom and three-dimensional (3D) electron paramagnetic resonance (EPR) images via the 6-spheres phantom. Specifically, two evaluations based on projection generation and image reconstruction were performed. For projection generation, evaluation was using a 2D disc phantom, the modified FORBILD phantom and the 6-spheres phantom. For image reconstruction, evaluations were performed using the FORBILD and 6-spheres phantom. During evaluation, 2 quantitative indices of root-mean-square-error (RMSE) and contrast-to-noise-ratio (CNR) were used., Results: Comparing to the use of ordinary pixel-driven projection method, RMSE of the SPLD based least-square algorithm was reduced from 0.0701 to 0.0384 and CNR was increased from 5.6 to 19.47 for 2D FORBILD phantom reconstruction. For 3D EPRI, RMSE of SPLD was also reduced from 0.0594 to 0.0498 and CNR was increased from 3.88 to 11.58. In addition, visual evaluation showed that images reconstructed in both 2D and 3D images suffered from high-frequency line-shape artifacts when using the ordinary pixel-driven projection method. However, using 3 new methods all suppressed the artifacts significantly and yielded more accurate reconstructions., Conclusions: Three proposed pixel-driven projection methods achieved more accurate iterative image reconstruction results. These new and more accurate methods can also be easily extended to other imaging modalities. Among them, SPLD method should be recommended to 3D and four dimensional (4D) EPR imaging.

Oxygen is the single most important molecule for sustaining life and, therefore, an important variable in tissue engineering and regenerative medicine. It has been shown that the change in oxygen concentration in an artificial or tissue-engineered graft affects cell survival, differentiation, and tissue growth in profound ways. However, at present, there are no reliable methods to map partial oxygen pressure (pO 2 ) in growing artificial tissues. Here, we adapt and test the suitability of electron paramagnetic resonance oxygen imaging (EPROI) in assessing tissue graft oxygenation in vitro. EPROI is an established method to assess absolute pO 2 and has been widely applied to study tumor hypoxia in small animals. In this study, we demonstrate the feasibility of EPROI in evaluating oxygen dynamics in tissue grafts. We measured oxygen concentration in mesenchymal stem cell (MSC)-seeded polylactic-co-glycolic acid (PLGA) scaffolds with variable porosity. The pO 2 maps of these scaffolds showed that the mean pO 2 inside the scaffolds was smaller than the ambient air pO 2 (21% oxygen, 160 torr) and was gradually increased with increasing pore size. We assessed the local oxygen dynamics of the MSC-seeded osteogenic scaffold made from collagen-chitosan hydrogels in a partially sealed Eppendorf tube. The change in pO 2 values as a function of time inside the graft showed that the cells had used available oxygen within first 2 h of the experiment and then went to a dormant low oxygen consumption state until the oxygen supply was reestablished. Collectively, these data suggest that EPROI could be successfully used for mapping pO 2 in tissue-engineered grafts. The knowledge of tissue graft oxygenation may be used to improve scaffold design and to assess the tissue viability and growth.

The optimization-based image reconstruction methods have been thoroughly investigated in the field of medical imaging. The Chambolle-Pock (CP) algorithm may be employed to solve these convex optimization image reconstruction programs. The preconditioned CP (PCP) algorithm has been shown to have much higher convergence rate than the ordinary CP (OCP) algorithm. This algorithm utilizes a preconditioner-parameter to tune the implementation of the algorithm to the specific application, which ranges from 0 and 2, but is often set to 1. In this work, we investigated the impact of the preconditioner-parameter on the convergence rate of the PCP algorithm when it is applied to the TV constrained, data-divergence minimization (TVDM) optimization based image reconstruction. We performed the investigations in the context of 2D computed tomography (CT) and 3D electron paramagnetic resonance imaging (EPRI). For 2D CT, we used the Shepp-Logan and two FORBILD phantoms. For 3D EPRI, we used a simulated 6-spheres phantom and a physical phantom. Study results showed that the optimal preconditioner-parameter depends on the specific imaging conditions. Simply setting the parameter equal to 1 cannot guarantee a fast convergence rate. Thus, this study suggests that one should adaptively tune the preconditioner-parameter to obtain the optimal convergence rate of the PCP algorithm.

Resonators for preclinical electron paramagnetic resonance imaging have been designed primarily for rodents and rabbits and have internal diameters between 16 and 51 mm. Lumped circuit resonators include loop-gap, Alderman-Grant, and saddle coil topologies and surface coils. Bimodal resonators are useful for isolating the detected signal from incident power and reducing dead time in pulse experiments. Resonators for continuous wave, rapid scan, and pulse experiments are described. Experience at the University of Chicago and University of Denver in design of resonators for in vivo imaging is summarized.

Radiation treatment success and high tumor oxygenation and success have been known to be highly correlated. This suggests that radiation therapy guided by images of tumor regions with low oxygenation, oxygen-guided radiation therapy (OGRT) may be a promising enhancement of cancer radiation treatment. Before applying the technique to human subjects, OGRT needs to be tested in animals, most easily in rodents. Electron paramagnetic resonance imaging provides quantitative maps of tissue and tumor oxygen in rodents with 1 mm spatial resolution and 1 torr pO 2 resolution at low oxygen levels. The difficulty of using mouse models is their small size and that of their tumors. To overcome this we used XRAD225Cx micro-CT/ therapy system and 3D printed conformal blocks. Radiation is delivered first to a uniform 15% tumor control dose for the whole tumor and then a boost dose to either hypoxic tumor regions or equal volumes of well oxygenated tumor. Delivery of the booster dose used a multiple beam angles to deliver radiation beams whose shape conforms to that of all hypoxic regions or fully avoids those regions. To treat/avoid all hypoxic regions we used individual radiation blocks 3D-printed from acrylonitrile butadiene styrene polymer infused with tungsten particles fabricated immediately after imaging to determine regions with pO 2 less than 10 torr. Preliminary results demonstrate the efficacy of the radiation treatment with hypoxic boosts with syngeneic FSa fibrosarcoma tumors in the legs of C3H mice.

A magnetometer designed for permanent magnet manufacturing and operated around 25 mT with 10ppm absolute accuracy is described. The magnetometer uses pulse electron paramagnetic resonance (EPR) methodology. The use of a pulsed broadband acquisition allowed reliable measurements in the presence of the magnetic field gradient and in relatively inhomogeneous magnetic fields of un-shimmed magnets.

The value of any measurement and a fortiori any measurement technology is defined by the reproducibility and the accuracy of the measurements. This implies a relative freedom of the measurement from factors confounding its accuracy. In the past, one of the reasons for the loss of focus on the importance of imaging oxygen in vivo was the difficulty in obtaining reproducible oxygen or pO 2 images free from confounding variation. This review will briefly consider principles of electron paramagnetic oxygen imaging and describe how it achieves absolute oxygen measurements. We will provide a summary review of the progress in biomedical EPR imaging, predominantly in cancer biology research, discuss EPR oxygen imaging for cancer treatment and tissue graft assessment for regenerative medicine applications., (Copyright © 2017. Published by Elsevier Inc.)

In rapid-scan EPR the magnetic field or frequency is repeatedly scanned through the spectrum at rates that are much faster than in conventional continuous wave EPR. The signal is directly-detected with a mixer at the source frequency. Rapid-scan EPR is particularly advantageous when the scan rate through resonance is fast relative to electron spin relaxation rates. In such scans, there may be oscillations on the trailing edge of the spectrum. These oscillations can be removed by mathematical deconvolution to recover the slow-scan absorption spectrum. In cases of inhomogeneous broadening, the oscillations may interfere destructively to the extent that they are not visible. The deconvolution can be used even when it is not required, so spectra can be obtained in which some portions of the spectrum are in the rapid-scan regime and some are not. The technology developed for rapid-scan EPR can be applied generally so long as spectra are obtained in the linear response region. The detection of the full spectrum in each scan, the ability to use higher microwave power without saturation, and the noise filtering inherent in coherent averaging results in substantial improvement in signal-to-noise relative to conventional continuous wave spectroscopy, which is particularly advantageous for low-frequency EPR imaging. This overview describes the principles of rapid-scan EPR and the hardware used to generate the spectra. Examples are provided of its application to imaging of nitroxide radicals, diradicals, and spin-trapped radicals at a Larmor frequency of ca. 250MHz., (Copyright © 2017 Elsevier Inc. All rights reserved.)

In vivo oximetry by pulsed electron paramagnetic resonance is based on measurements of changes in electron spin relaxation rates of probe molecules, such as the triarylmethyl radicals. A series of experiments was performed at frequencies between 250 MHz and 1.5 GHz to assist in the selection of an optimum frequency for oximetry. Electron spin relaxation rates for the triarylmethyl radical OX063 as a function of radical concentration, salt concentration, and resonance frequency were measured by electron spin echo 2-pulse decay and 3-pulse inversion recovery in the frequency range of 250 MHz-1.5 GHz. At constant OX063 concentration, 1/T 1 decreases with increasing frequency because the tumbling dependent processes that dominate relaxation at 250 MHz are less effective at higher frequency. 1/T 2 also decreases with increasing frequency because 1/T 1 is a significant contribution to 1/T 2 for trityl radicals in fluid solution. 1/T 2 -1/T 1 , the incomplete motional averaging contribution to 1/T 2 , increases with increasing frequency. At constant frequency, relaxation rates increase with increasing radical concentration due to contributions from collisions that are more effective for 1/T 2 than 1/T 1 . The collisional contribution to relaxation increases as the concentration of counter-ions in solution increases, which is attributed to interactions of cations with the negatively charged radicals that decrease repulsion between trityl radicals. The Signal-to-Noise ratio (S/N) of field-swept echo-detected spectra of OX063 were measured in the frequency range of 400 MHz-1 GHz. S/N values, normalized by √Q, increase as frequency increases. Adding salt to the radical solution decreased S/N because salt lowers the resonator Q. Changing the temperature from 19 to 37 °C caused little change in S/N at 700 MHz. Both slower relaxation rates and higher S/N at higher frequencies are advantageous for oximetry. The potential disadvantage of higher frequencies is the decreased depth of penetration into tissue.

This study investigated the location and distribution of paramagnetic species in apple seeds using electron paramagnetic resonance (EPR) and X-band (9 GHz) EPR imaging (EPRI). EPR primarily detected two paramagnetic species per measured seed. These two different radical species were assigned as stable radicals and Mn 2+ species based on the g values and hyperfine components. The signal from the stable radical was noted at g ≈ 2.00 and was strong and relatively stable. The subsequent noninvasive EPRI of the radical present in each seed revealed that the stable radicals were located primarily in the seed coat, with very few radicals observed in the cotyledon of the seed. These results indicate that the stable radical species were only found within the seed coat, and few radical species were found in other seed parts.

Thiol redox status is an important physiologic parameter that affects the success or failure of cancer treatment. Rapid scan electron paramagnetic resonance (RS EPR) is a novel technique that has shown higher signal-to-noise ratio than conventional continuous-wave EPR in in vitro studies. Here we used RS EPR to acquire rapid three-dimensional images of the thiol redox status of tumors in living mice. This work presents, for the first time, in vivo RS EPR images of the kinetics of the reaction of 2 H, 15 N-substituted disulfide-linked dinitroxide (PxSSPx) spin probe with intracellular glutathione. The cleavage rate is proportional to the intracellular glutathione concentration. Feasibility was demonstrated in a FSa fibrosarcoma tumor model in C3H mice. Similar to other in vivo and cell model studies, decreasing intracellular glutathione concentration by treating mice with l-buthionine sulfoximine (BSO) markedly altered the kinetic images., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Rapid expansion of tumor cells that outpace existing vasculature essential for nutrient and oxygen support as well as waste removal, correlates with profound changes in the microenvironment including angiogenesis, vasodilation, glucose metabolism, and cell cycle perturbations. Since hypoxic cells are up to three times more radioresistant than normoxic cells, identification of hypoxic populations to predict radiotherapeutic outcome is important. The consequences of hypoxia and activated proteins contribute to radioresistant tumors and radiotherapeutic failure. Stereotactic MCa4 tumor tissue biopsies from mouse tumors that were guided by electron paramagnetic resonance (EPR) O 2 imaging were examined for hypoxia-induced proteins. The oxygen broadening of narrow EPR spectral lines or, equivalently, the increase in relaxation rates of electron magnetization, report pO 2 with 1-2 torr resolution in image voxels less than 1 mm 3 . The pO 2 reporter molecule OX063d64 (trityl) was used to acquire the data described here. Trityl appears to be selectively retained in tumors with a half-life of ~30 min. We used an inversion recovery electron spin echo (IRESE) to measure the T1 rate of the trityl inside the tumor bearing leg. We estimate our uncertainty in pO 2 measurement to be 1-3 torr per voxel. Three hypoxic cell biomarkers, hypoxic-induced factor 1-alpha (HIF-1α), vascular endothelial growth factor (VEGF), and carbonic anhydrase IX (CA9), were examined using the ELISA assay. Quantification of these proteins based on results from the ELISA immunoassay kits indicate a strong correlation between EPR pO 2 -identified hypoxic fractions (<10 torr) and HIF-1α, VEGF, and CA9. We clearly demonstrate that hypoxic regions in tumors generate substantial amounts of HIF- 1α, VEGF, and CA9 protein.

Modern standards for radiation treatment do not take into account tumor oxygenation for radiation treatment planning. Strong correlation between tumor oxygenation and radiation treatment success suggests that oxygen-guided radiation therapy (OGRT) may be a promising enhancement of cancer radiation treatment. We have developed an OGRT protocol for rodents. Electron paramagnetic resonance (EPR) imaging is used for recording oxygen maps with high spatial resolution and excellent accuracy better than 1 torr. Radiation is delivered with an animal intensity modulated radiation therapy (IMRT) XRAD225Cx micro-CT/ therapy system. The radiation plan is delivered in two steps. First, a uniform 15% tumor control dose (TCD 15 ) is delivered to the whole tumor. In the second step, an additional booster dose amounting to the difference between TCD 98 and TCD 15 is delivered to radio-resistant, hypoxic tumor regions. Delivery of the booster dose is performed using a multiport conformal beam protocol. For radiation beam shaping we used individual radiation blocks 3D-printed from tungsten infused ABS polymer. Calculation of beam geometry and the production of blocks is performed next to the EPR imager, immediately after oxygen imaging. Preliminary results demonstrate the sub-millimeter precision of the radiation delivery and high dose accuracy. The efficacy of the radiation treatment is currently being tested on syngeneic FSa fibrosarcoma tumors grown in the legs of C3H mice.

The triarylmethyl radical OX063d24 is currently used for pulsed electron paramagnetic resonance oximetry at 250 MHz. Both 1/T 1 and 1/T 2 increase with increasing oxygen concentration. The dependence of 1/T 1 on probe concentration is smaller than for 1/T 2 . To inform the selection of the optimum frequency for in vivo oximetry 1/T 1 , 1/T 2 and signal-to-noise were measured as a function of frequency between 400 and 1000 MHz on a variable-frequency spectrometer with an adjustable-frequency cross-loop resonator. 1/T 1 and 1/T 2 decrease with increasing frequency and signal-to-noise increases with increasing frequency, which are all favourable for imaging at higher frequencies. However, depth of penetration of the radio frequency (RF) into an animal decreases with increasing frequency. Assuming that the RF loss in the animal to be studied determines the resonator Q, our results indicate that the optimum frequency for in vivo imaging will be determined by the desired depth of penetration in the tissue.

In pulsed magnetic resonance, the excitation power is many orders of magnitude larger than that induced by the spin system in the receiving coil or resonator. The receiver must be protected during and immediately after the excitation pulse to allow for the energy stored in the resonator to dissipate to a safe level. The time during which the signal is not detected, the instrumental dead-time, can be shortened by using magnetically decoupled excitation and receive coils. Such coils are oriented, with respect to each other, in a way that minimizes the total magnetic flux produced by one coil in the other. We suggest that magnetically decoupled coils can be isolated to a larger degree by tuning them to separate frequencies. Spins are excited at one frequency, and the echo signal is detected at another. Sinusoidal magnetic field modulation that rapidly changes the Larmor frequency of the spins between the excitation and detection events is used to ensure the resonance conditions for both coils. In this study, the relaxation times of trityl-CD3 were measured in a field-modulated pulsed EPR experiment and compared to results obtained using a standard spin echo method. The excitation and receive coils were tuned to 245 and 256.7MHz, respectively. Using an available rapid-scan, cross-loop EPR resonator, we demonstrated an isolation improvement of approximately 20-30dB due to frequency decoupling. Theoretical analysis, numerical simulations, and proof-of-concept experiments demonstrated that substantial excitation-detection decoupling can be achieved. A pulsed L-band system, including a small volume bi-modal resonator equipped with modulation coils, was constructed to demonstrate fivefold dead-time reduction in comparison with the standard EPR experiment. This was achieved by detuning of the excitation and receive coils by 26MHz and using sinusoidal modulation at 480kHz., (Copyright © 2016 Elsevier Inc. All rights reserved.)