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Electron paramagnetic resonance (EPR) spectroscopy using oxygen-sensing implants can provide reliable and repeated measurements of the partial pressure of oxygen (pO 2 ) over a period of months or longer; however, it does not provide accurate information about the distribution of tissue oxygenation. While EPR imaging has the capability to provide spatially resolved oxygen data, it is time-consuming and not optimized for discrete number of implants. Previous reports suggest multi-site algorithms, which would require either the implants to be aligned in a certain way so as to deconvolve them using a linear magnetic field gradient or sparse imaging of the implants from a small number of 3D projections. In this paper, we present a simpler and much faster method to estimate the pO 2 histogram from a composite, single-scan EPR spectrum measured without applying field gradients to separate the EPR signals from multiple randomly placed oxygen-sensing implants. The method is optimized for a discrete number of implants, validated using simulations, experimental phantoms and in animal models. The results established the composite spectral fitting algorithm as a reliable and robust tool for multi-site oximetry.

Objectives/hypothesis: To determine if electron paramagnetic resonance (EPR) oximetry is a viable technology to aid in flap monitoring., Study Design: Prospective cohort., Methods: This was a cohort study assessing accuracy and speed of EPR oximetry in detecting ischemia of a saphenous artery-based flap in a rat model, using transcutaneous oximetry as a control. Measurements were obtained under both resting and ischemic conditions for nine Sprague Dawley rats (18 flaps), for 3 postoperative days following flap elevation., Results: The mean partial pressure of oxygen prior to tourniquet application was 66.9 ± 8.9 mm Hg with EPR oximetry and 64.7 ± 5.2 mm Hg with transcutaneous oximetry (P = .45). Mean partial pressures of oxygen during tourniquet application were 8.9 ± 3.2 mm Hg and 8.5 ± 2.9 mm Hg for EPR oximetry and transcutaneous oximetry, respectively (P = .48), and 67.2 ± 6.9 mm Hg and 65.3 ± 6.1 mm Hg after tourniquet release for EPR oximetry and transcutaneous oximetry, respectively (P = .44). The mean ischemia detection time of EPR oximetry was 49 ± 21 seconds., Conclusions: Offering timely, accurate, and noninvasive tissue oxygen measurements, EPR oximetry is a promising adjunct in flap monitoring., Level of Evidence: NA Laryngoscope, 129:E415-E419, 2019., (© 2019 The American Laryngological, Rhinological and Otological Society, Inc.)

Electron spin resonance (ESR) spectroscopy of paramagnetic species in single crystals is a powerful tool for characterizing the latter's magnetic interaction parameters in detail. Conventional ESR systems are optimized for millimeter-size samples and make use of cavities and resonators that accommodate tubes and capillaries in the range 1-5 mm. Unfortunately, in the case of many interesting materials such as enzymes and inorganic catalytic materials (e.g., zeolites), single crystals can only be obtained in micron-scale sizes (1-200 µ m). To boost ESR sensitivity and to enable experiments on microcrystals, the ESR resonator needs to be adapted to the size and shape of these specific samples. Here, we present a unique family of miniature surface resonators, known as "ParPar" resonators, whose mode volume and shape are optimized for such micron-scale single crystals. This approach significantly improves upon the samples' filling factor and thus enables the measurement of much smaller crystals than was previously possible. We present here the design of such resonators with a typical mode dimension of 20-50 µ m, as well as details about their fabrication and testing methods. The devices' resonant mode(s) are characterized by ESR microimaging and compared to the theoretical calculations. Moreover, experimental ESR spectra of single microcrystals with typical sizes of ∼25-50 µ m are presented. The measured spin sensitivity for the 50- µ m resonator at cryogenic temperatures of 50 K is found to be ∼1.8 × 10 6 spins/G √Hz for a Cu-doped single crystal sample that is representative of many biological samples of relevance.

Electron spin resonance (ESR) is a powerful analytical technique used for the detection, quantification, and characterization of paramagnetic species ranging from stable organic free radicals and defects in crystals to gaseous oxygen. Traditionally, ESR requires the use of complex instrumentation, including a large magnet and a microwave resonator in which the sample is placed. Here, we present an alternative to the existing approach by inverting the typical measurement topology, namely placing the ESR magnet and resonator inside the sample rather than the other way around. This new development relies on a novel self-contained ESR sensor with a diameter of just 2 mm and length of 3.6 mm, which includes both a small permanent magnet assembly and a tiny (∼1 mm in size) resonator for spin excitation and detection at a frequency of ∼2.6 GHz. The spin sensitivity of the sensor has been measured to be ∼10 11 spins/√Hz, and its concentration sensitivity is ∼0.1 mM, using reference samples with a measured volume of just ∼10 nL. Our new approach can be applied for monitoring the partial pressure of oxygen in vitro and in vivo through its paramagnetic interaction with another stable radical, as well as for simple online quantitative inspection of free radicals generated in reaction vessels and electrochemical cells via chemical processes.

Cerebral tissue oxygenation (oxygen tension, pO 2 ) is a critical parameter that is closely linked to brain metabolism, function, and pathophysiology. In this work, we have used electron paramagnetic resonance oximetry with a deep-tissue multi-site oxygen-sensing probe, called implantable resonator, to monitor temporal changes in cerebral pO 2 simultaneously at four sites in a rabbit model of ischemic stroke induced by embolic clot. The pO 2 values in healthy brain were not significantly different among the four sites measured over a period of 4 weeks. During exposure to 15% O 2 (hypoxia), a sudden and significant decrease in pO 2 was observed in all four sites. On the other hand, brief exposure to breathing carbogen gas (95% O 2  + 5% CO 2 ) showed a significant increase in the cerebral pO 2 from baseline value. During ischemic stroke, induced by embolic clot in the left brain, a significant decline in the pO 2 of the left cortex (ischemic core) was observed without any change in the contralateral sites. While the pO 2 in the non-infarct regions returned to baseline at 24-h post-stroke, pO 2 in the infarct core was consistently lower compared to the baseline and other regions of the brain. The results demonstrated that electron paramagnetic resonance oximetry with the implantable resonator can repeatedly and simultaneously report temporal changes in cerebral pO 2 at multiple sites. This oximetry approach can be used to develop interventions to rescue hypoxic/ischemic tissue by modulating cerebral pO 2 during hypoxic and stroke injury.

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.

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized clinically by cognitive decline and memory loss. The pathological features are amyloid-β peptide (Aβ) plaques and intracellular neurofibrillary tangles. Many studies have suggested that oxidative damage induced by reactive oxygen species (ROS) is an important mechanism for AD progression. Our recent study demonstrated that oxidative stress could further impair mitochondrial function. In the present study, we adopted a transgenic mouse model of AD (mAPP, overexpressing AβPP/Aβ in neurons) and performed redox measurements using in vivo electron paramagnetic resonance (EPR) imaging with methoxycarbamyl-proxyl (MCP) as a redox-sensitive probe for studying oxidative stress in an early stage of pathology in a transgenic AD mouse model. Through assessing oxidative stress, mitochondrial function and cognitive behaviors of mAPP mice at the age of 8-9 months, we found that oxidative stress and mitochondrial dysfunction appeared in the early onset of AD. Increased ROS levels were associated with defects of mitochondrial and cognitive dysfunction. Notably, the in vivo EPR method offers a unique way of assessing tissue oxidative stress in living animals under noninvasive conditions, and thus holds a potential for early diagnosis and monitoring the progression of AD.

The first systematic multi-center study of the clinical use of EPR oximetry has begun, with funding as a PPG from the NCI. Using particulate oxygen sensitive EPR, materials in three complementary forms (India Ink, "OxyChips", and implantable resonators) the clinical value of the technique will be evaluated. The aims include using repeated measurement of tumor pO2 to monitor the effects of treatments on tumor pO2, to use the measurements to select suitable subjects for the type of treatment including the use of hyperoxic techniques, and to provide data that will enable existing clinical techniques which provide data relevant to tumor pO2 but which cannot directly measure it to be enhanced by determining circumstances where they can give dependable information about tumor pO2.

Oxygen (O2) plays a central role in most living organisms. The concentration of O2 is important in physiology and pathology. Despite the importance of accurate knowledge of the O2 levels, there is very limited capability to measure with high spatial resolution its distribution in millimeter-scale live biological samples. Many of the current oximetric methods, such as oxygen microelectrodes and fluorescence lifetime imaging, are compromised by O2 consumption, sample destruction, invasiveness, and difficulty to calibrate. Here, we present a new method, based on the use of the pulsed electron spin resonance (ESR) microimaging technique to obtain a 3D mapping of oxygen concentration in millimeter-scale biological samples. ESR imaging requires the incorporation of a suitable stable and inert paramagnetic spin probe into the desirable object. In this work, we use microcrystals of a paramagnetic spin probe in a new crystallographic packing form (denoted tg-LiNc-BuO). These paramagnetic species interact with paramagnetic oxygen molecules, causing a spectral line broadening that is linearly proportional to the oxygen concentration. Typical ESR results include 4D spatial-spectral images that give an indication about the oxygen concentration in different regions of the sample. This new oximetry microimaging method addresses all the problems mentioned above. It is noninvasive, sensitive to physiological oxygen levels, and easy to calibrate. Furthermore, in principle, it can be used for repetitive measurements without causing cell damage. The tissue model used in this research is spheroids of Human Colorectal carcinoma cell line (HCT-116) with a typical diameter of ∼600μm. Most studies of the microenvironmental O2 conditions inside such viable spheroids carried out in the past used microelectrodes, which require an invasive puncturing of the spheroid and are also not applicable to 3D O2 imaging. High resolution 3D oxygen maps could make it possible to evaluate the relationship between morphological and physiological alterations in the spheroids, which would help understand the oxygen metabolism in solid tumors and its correlation with the susceptibility of tumors to various oncologic treatments., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Low level of oxygen (hypoxia) is a critical factor that defines the pathological consequence of several pathophysiologies, particularly ischemia, that usually occur following the blockage of a blood vessel in vital organs, such as brain and heart, or abnormalities in the microvasculature, such as peripheral vascular disease. Therefore, methods that can directly and repeatedly quantify oxygen levels in the brain and heart will significantly improve our understanding of ischemic pathologies. Importantly, such oximetry capability will facilitate the development of strategies to counteract low levels of oxygen and thereby improve outcome following stroke or myocardial infarction. In vivo electron paramagnetic resonance (EPR) oximetry has the capability to monitor tissue oxygen levels in real time. The method has largely been tested and used in experimental animals, although some clinical measurements have been performed. In this chapter, a brief overview of the methodology to repeatedly quantify oxygen levels in the brain and heart of experimental animal models, ranging from mice to swine, is presented. EPR oximetry requires a one-time placement of an oxygen-sensitive probe in the tissue of interest, while the rest of the procedure for reliable, accurate, and repeated measurements of pO2 (partial pressure of oxygen) is noninvasive and can be repeated as often as desired. A multisite oximetry approach can be used to monitor pO2 at many sites simultaneously. Building on significant advances in the application of EPR oximetry in experimental animal models, spectrometers have been developed for use in human subjects. Initial feasibility of pO2 measurement in solid tumors of patients has been successfully demonstrated., (© 2015 Elsevier Inc. All rights reserved.)

Electron paramagnetic resonance (EPR) spectroscopy has been well established as a viable technique for measurement of free radicals and oxygen in biological systems, from in vitro cellular systems to in vivo small animal models of disease. However, the use of EPR in human subjects in the clinical setting, although attractive for a variety of important applications such as oxygen measurement, is challenged with several factors including the need for instrumentation customized for human subjects, probe, and regulatory constraints. This article describes the rationale and development of the first clinical EPR systems for two important clinical applications, namely, measurement of tissue oxygen (oximetry) and radiation dose (dosimetry) in humans. The clinical spectrometers operate at 1.2 GHz frequency and use surface-loop resonators capable of providing topical measurements up to 1 cm depth in tissues. Tissue pO2 measurements can be carried out noninvasively and repeatedly after placement of an oxygen-sensitive paramagnetic material (currently India ink) at the site of interest. Our EPR dosimetry system is capable of measuring radiation-induced free radicals in the tooth of irradiated human subjects to determine the exposure dose. These developments offer potential opportunities for clinical dosimetry and oximetry, which include guiding therapy for individual patients with tumors or vascular disease by monitoring of tissue oxygenation. Further work is in progress to translate this unique technology to routine clinical practice., (Copyright © 2014 AUR. Published by Elsevier Inc. All rights reserved.)

The feasibility of EPR oximetry using a single-probe implantable oxygen sensor (ImOS) was tested for repeated measurement of pO₂ in skeletal muscle and ectopic 9L tumors in rats. The ImOS (50 mm length) were constructed using nickel-chromium alloy wires, with lithium phthalocyanine (LiPc, oximetry probe) crystals loaded in the sensor loop and coated with AF 2400(®) Teflon. These ImOS were implanted into the skeletal muscle in the thigh and subcutaneous 9L tumors. Dynamic changes in tissue pO₂ were assessed by EPR oximetry at baseline, during tumor growth, and repeated hyperoxygenation with carbogen breathing. The mean skeletal muscle pO₂ of normal rats was stable and significantly increased during carbogen inhalation in experiments repeated for 12 weeks. The 9L tumors were hypoxic with a tissue pO₂ of 12.8 ± 6.4 mmHg on day 1; however, the response to carbogen inhalation varied among the animals. A significant increase in the glioma pO₂ was observed during carbogen inhalation on day 9 and day 14 only. In summary, EPR oximetry with ImOS allowed direct and longitudinal oxygen measurements in deep muscle tissue and tumors. The heterogeneity of 9L tumors in response to carbogen highlights the need to repeatedly monitor pO₂ to confirm tumor oxygenation so that such changes can be taken into account in planning therapies and interpreting results.

The use of electron paramagnetic resonance (EPR) oximetry for oxygen measurements in deep tissues (>1 cm) is challenging due to the limited penetration depth of the microwave energy. To overcome this limitation, implantable resonators, having a small (0.5 mm diameter) sensory loop containing the oxygen-sensing paramagnetic material connected by a pair of twisted copper wire to a coupling loop (8-10 mm diameter), have been developed, which enable repeated measurements of deep-tissue oxygen levels (pO2, partial pressure of oxygen) in the brain and tumors of rodents. In this study, we have demonstrated the feasibility of measuring dynamic changes in pO2 in the heart and lung of rats using deep-tissue implantable oxygen sensors. The sensory loop of the resonator contained lithium octa-n-butoxynaphthalocyanine (LiNc-BuO) crystals embedded in polydimethylsiloxane (PDMS) polymer and was implanted in the myocardial tissue or lung pleura. The external coupling loop was secured subcutaneously above chest. The rats were exposed to different breathing gas mixtures while undergoing EPR measurements. The results demonstrated that implantable oxygen sensors provide reliable measurements of pO2 in deep tissues such as heart and lung under adverse conditions of cardiac and respiratory motions.

EPR oximetry, which enables reliable, accurate, and repeated measurements of the partial pressure of oxygen in tissues, provides a unique opportunity to investigate the role of oxygen in the pathogenesis and treatment of several diseases including cancer, stroke, and heart failure. Building on significant advances in the in vivo application of EPR oximetry for small animal models of disease, we are developing suitable probes and instrumentation required for use in human subjects. Our laboratory has established the feasibility of clinical EPR oximetry in cancer patients using India ink, the only material presently approved for clinical use. We now are developing the next generation of probes, which are both superior in terms of oxygen sensitivity and biocompatibility including an excellent safety profile for use in humans. Further advances include the development of implantable oxygen sensors linked to an external coupling loop for measurements of deep-tissue oxygenations at any depth, overcoming the current limitation of 10 mm. This paper presents an overview of recent developments in our ability to make meaningful measurements of oxygen partial pressures in human subjects under clinical settings.

The detection, quantification, and imaging of short-lived reactive oxygen species, such as superoxide, in live biological specimens have always been challenging and controversial. Fluorescence-based methods are nonspecific, and electron spin resonance (ESR) spin-trapping methods require high probe concentrations and lack the capability for sufficient image resolution. In this work, a novel (to our knowledge), sensitive, small ESR imaging resonator was used together with a stable spin probe that specifically reacts with superoxide with a high reaction rate constant. This ESR spin-probe-based methodology was used to examine superoxide generated in a plant root as a result of an apical leaf injury. The results show that the spin probe rapidly permeated the plant's extracellular space. Upon injury of the plant tissue, superoxide was produced and the ESR signal decreased rapidly in the injured parts as well as in the distal part of the root. This is attributed to superoxide production and thus provides a means of quantifying the level of superoxide in the plant. The spin probe's narrow single-line ESR spectrum, together with the sensitive imaging resonator, facilitates the quantitative measurement of superoxide in small biological samples, such as the plant's root, as well as one-dimensional imaging along the length of the root. This type of methodology can be used to resolve many questions involving the production of apoplastic superoxide in plant biology., (Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Acute and chronic intestinal ischemia has been linked to the development of gastrointestinal symptoms such as abdominal pain, nausea and vomiting, bowel dysfunction and more seriously, the complications of sepsis, shock and death. Advances in electron paramagnetic resonance (EPR) oximetry have resulted in accurate and reliable in vivo measurement of the partial pressure of oxygen (pO(2)) in solid organs (e.g., muscle, heart) [1], but has yet to be tested in thin walled organs such as intestine. Our ultimate goal is to noninvasively monitor intestinal pO(2) during acute and chronic intestinal ischemia in a rat model. A series of experiments to deliver oxygen-sensitive indicator probes to the small/large intestine by intravenous, luminal and wall injection, as well as direct placement of a solid probe against the outer intestinal wall were attempted. Only the LiNc:BuO:PDMS chip sutured to the peritoneal wall and in direct contact with the intestine allowed for noninvasive pO(2) measurement by EPR. However, the validity of site-specific intestinal pO(2) measurement could not be confirmed and the obtained pO(2) value likely reflected peritoneal cavity oxygenation. Developing methods for probe placement on or inside the intestinal wall are needed for noninvasive, site-specific intestinal pO(2) measurement by EPR to track changes during acute and chronic intestinal ischemia.

A new paramagnetic crystalline material, namely, lithium naphthalocyanine (LiNc), whose electron-paramagnetic-resonance (EPR) line width is highly sensitive to oxygen content, has been evaluated for use as oximetry probe in cells and tissues. Previously,we reported on the synthesis, structural framework,magnetic and oxygen-sensing properties of LiNc microcrystalline powder (Pandian et al, J. Mater. Chem. 19, 4138, 2009). The material exhibited a single, sharp EPR line that showed a highly linear response of its width to surrounding molecular oxygen (pO(2)) with a sensitivity of 31.2 mG/mmHg. In the present study, we evaluated the suitability of this material for in vivo oximetry in biological systems. We observed that the probe was stable in tissues for more than two months without any adverse effect on its oxygen-sensing properties. We further demonstrated that the probe can be prepared in sub-micron sizes for uptake by stem cells. Thus, the high oxygen sensitivity, biocompatibility, and long-term stability in tissues may be useful for high-resolution EPR oximetry.

Electron paramagnetic resonance (EPR) oximetry is a powerful technique capable of providing accurate, reliable, and repeated measurements of tissue oxygenation, which is crucial to the diagnosis and treatment of several pathophysiological conditions. Measurement of tissue pO(2) by EPR involves the use of paramagnetic, oxygen-sensitive probes, which can be either soluble (molecular) in nature or insoluble paramagnetic materials. Development of innovative strategies to enhance the biocompatibility and in vivo application of these oxygen-sensing probes is crucial to the growth and clinical applicability of EPR oximetry. Recent research efforts have aimed at encapsulating particulate probes in bioinert polymers for the development of biocompatible EPR probes. In this study, we have developed novel EPR oximetry probes, called perchlorotriphenylmethyl triester (PTM-TE):polydimethyl siloxane (PDMS) chips, by dissolving and incorporating the soluble (molecular) EPR probe, PTM-TE, in an oxygen-permeable polymer matrix, PDMS. We demonstrate that such incorporation (doping) of PTM-TE in PDMS enhanced its oxygen sensitivity several fold. The cast-molding method of fabricating chips enabled them to be made with increasing amounts of PTM-TE (spin density). Characterization of the spin distribution within the PDMS matrix, using EPR micro-imaging, revealed potential inhomogeneties, albeit with no adverse effect on the oxygen-sensing characteristics of PTM-TE:PDMS. The chips were resistant to autoclaving or in vitro oxidoreductant treatment, thus exhibiting excellent in vitro biostability. Our results establish PTM-TE:PDMS as a viable probe for biological oxygen-sensing, and also validate the incorporation of soluble probes in polymer matrices as an innovative approach to the development of novel probes for EPR oximetry., (Copyright 2010 Elsevier B.V. All rights reserved.)

Lithium naphthalocyanine (LiNc) is a crystalline material that has significant potential as a probe for EPR (electron paramagnetic resonance)-based biological oximetry (Pandian et al. J. Mater. Chem. 19:4138-4147, 2009a). However, implantation of LiNc crystals in tissues in raw or neat form is undesirable since dispersion of crystals in tissue may lead to loss of EPR signal, while also exacerbating biocompatibility concerns due to tissue exposure. To overcome these concerns, we have encapsulated LiNc crystals in an oxygen-permeable polymer, Teflon AF 2400 (TAF). Fabrication of TAF films incorporating LiNc particles (denoted as LiNc:TAF chip) was carried out using solvent-evaporation techniques. The EPR linewidth of LiNc:TAF chip was linearly dependent on oxygen-partial pressure (pO(2)) and did not change significantly relative to neat LiNc crystals. LiNc:TAF chip responded to changes in pO(2) reproducibly, enabling dynamic measurements of oxygenation in real time. The LiNc:TAF chips were stable in tissues for more than 2 months and were capable of providing repeated measurements of tissue oxygenation for extended periods of time. The results demonstrated that the newly fabricated, highly oxygen-sensitive LiNc:TAF chip will enhance the applicability of EPR oximetry for long-term and clinical applications.

It is widely accepted that redox status, along with the partial pressure of oxygen (pO(2)), determines the efficacy of some therapeutic methods applied to treat tumors, including radiation. Redox status, evaluated by the reduction of a nitroxyl probe, was reportedly heterogeneous in a mouse tumor model. However, neither variation of heterogeneity of the redox status among mice nor the relation of the redox status to pO(2) in tumors has been characterized sufficiently. In this study, the regional reduction status in a mouse radiation-induced fibrosarcoma tumor model was evaluated using sequential three-dimensional electron paramagnetic resonance (EPR) imaging after i.v. injection of a tissue-permeable nitroxyl probe, HM-PROXYL. The regional decay of HM-PROXYL signal obeyed first-order kinetics, and the amplitude of the reduction rate and extent of its heterogeneity in a tumor varied among six mice. The tissue pO(2) was measured using EPR oximetry with lithium phthalocyanine (LiPc) microcrystals implanted within the tumor. The location of LiPc was determined with EPR imaging. A sequential image was obtained following the injection of HM-PROXYL, even after LiPc implantation, by choosing an HM-PROXYL signal peak which does not overlap with the signal of LiPc. The relationship between pO(2) and the reduction rate at the region of pO(2) measurement was found to be low (r = 0.357) in 13 tumor-bearing mice, indicating that the extent of oxygenation does not necessarily affect the redox status under air-breathing conditions. The results strongly indicate the necessity of measurements of both redox status and oxygenation in every tumor to characterize tumor physiology., ((c)2010 AACR.)

Lithium naphthalocyanine (LiNc) is a microcrystalline EPR oximetry probe with high sensitivity to oxygen [R.P. Pandian, M. Dolgos, C. Marginean, P.M. Woodward, P.C. Hammel, P.T. Manoharan, P. Kuppusamy, Molecular packing and magnetic properties of lithium naphthalocyanine crystal: hollow channels enabling permeability and paramagnetic sensitivity to molecular oxygen J. Mater. Chem. 19 (2009) 4138-4147]. However, direct implantation of the crystals in the tissue for in vivo oxygen measurements may be hindered by concerns associated with their direct contact with the tissue/cells and loss of EPR signal due to particle migration in the tissue. In order to address these concerns, we have developed encapsulations (chips) of LiNc microcrystals in polydimethyl siloxane (PDMS), an oxygen-permeable, bioinert polymer. Oximetry evaluation of the fabricated chips revealed that the oxygen sensitivity of the crystals was unaffected by encapsulation in PDMS. Chips were stable against sterilization procedures or treatment with common biological oxidoreductants. In vivo oxygen measurements established the ability of the chips to provide reliable and repeated measurements of tissue oxygenation. This study establishes PDMS-encapsulated LiNc as a potential probe for long-term and repeated measurements of tissue oxygenation., (Copyright (c) 2009 Elsevier Inc. All rights reserved.)

Microcrystals of lithium octa-n-butoxynaphthalocyanine (LiNc-BuO) in a bio-compatible and oxygen-permeable polymer matrix of poly-dimethyl-siloxane (PDMS) can be used for repetitive non-invasive imaging of oxygen in live specimens by means of mm-scale electron spin resonance (ESR) imaging. This probe denoted as "oxychip" was characterized by high-resolution mum-scale ESR microcopy to reveal the fine details of its spatial and spectral properties. The ESR micro-images of a typical oxychip device showed that while the spatial distribution of the microcrystals in the polymer is fairly homogenous (as revealed by optical microscopy), the ESR signal originates only from a very few dominant crystals. Furthermore, spectral-spatial analysis in a microcrystal and a sub-microcrystal spatial resolution reveals that each crystal has a slightly different g-factor and also exhibits variations in linewidth, possibly due to the slightly different individual crystallization process., (Copyright (c) 2009 Elsevier Inc. All rights reserved.)

Free radicals are highly reactive compounds that play an essential role in many biological processes, both beneficial and deleterious. Detection and quantification of these species is critical to develop a better understanding of normal and pathophysiological functions at the cellular and tissue levels. Electron paramagnetic resonance (EPR) spectroscopy is the technique most commonly used for this purpose through the detection of exogenous probes or spin traps that interact with the free radical species of interest. Over the past several years, the spatial and temporal distribution of free radicals within cells and tissues has been of particular interest. This chapter briefly explains the principles and challenges in the use of EPR for biological samples and introduces the concept of EPR for free radical imaging purposes. In addition, specific examples are given for the use of EPR imaging in four principal areas: free radical probes, nitric oxide (NO), redox state, and oxygen (O(2)) concentration.

Lithium octa-n-butoxynaphthalocyanine (LiNc-BuO) is a promising probe for biological electron paramagnetic resonance (EPR) oximetry and is being developed for clinical use. However, clinical applicability of LiNc-BuO may be hindered by potential limitations associated with biocompatibility, biodegradation, and migration of individual crystals in tissue. To overcome these limitations, we have encapsulated LiNc-BuO crystals in polydimethyl siloxane (PDMS), an oxygen-permeable and bioinert polymer, to fabricate conveniently implantable and retrievable oxygen-sensing chips. Encapsulation was performed by a simple cast-molding process, giving appreciable control over size, shape, thickness and spin density of chips. The in vitro oxygen response of the chip was linear, reproducible, and not significantly different from that of unencapsulated crystals. Cast-molding of the structurally-flexible PDMS enabled the fabrication of chips with tailored spin densities, and ensured non-exposure of embedded LiNc-BuO, mitigating potential biocompatibility/toxicological concerns. Our results establish PDMS-encapsulated LiNc-BuO as a promising candidate for further biological evaluation and potential clinical application.

The use of oxygen-sensing water-insoluble paramagnetic probes, such as lithium octa-n-butoxynaphthalocyanine (LiNc-BuO), enables repeated measurements of pO(2) from the same location in tissue by electron paramagnetic resonance (EPR) spectroscopy. In order to facilitate direct in vivo application, and hence eventual clinical applicability, of LiNc-BuO, we encapsulated LiNc-BuO microcrystals in polydimethylsiloxane (PDMS), an oxygen-permeable and bioinert polymer, and developed an implantable chip. In vitro evaluation of the chip, performed under conditions of sterilization, high-energy irradiation, and exposure to cultured cells, revealed that it is biostable and biocompatible. Implantation of the chip in the gastrocnemius muscle tissue of mice showed that it is capable of repeated and real-time measurements of tissue oxygenation for an extended period. Functional evaluation using a murine tumor model established the suitability and applicability of the chip for monitoring tumor oxygenation. This study establishes PDMS-encapsulated LiNc-BuO as a promising choice of probe for clinical EPR oximetry.

Crystalline lithium phthalocyanine (LiPc) can be used to sense oxygen. To enhance biocompatibility/stability of LiPc, we encapsulated LiPc in Teflon AF (TAF), cellulose acetate (CA), and polyvinyl acetate (PVAc) (TAF, previously used to encapsulate LiPc, was a comparator). We identified water-miscible solvents that don't dissolve LiPc crystals, but are solvents for the polymers, and encapsulated crystals by solvent evaporation. Oxygen sensitivity of films was characterized in vitro and in vivo. Encapsulation did not change LiPc oximetry properties in vitro at anoxic conditions or varying partial pressures of oxygen (pO2). EPR linewidth of encapsulated particles was linear with pO2, responding to pO2 changes quickly and reproducibly for dynamic measurements. Encapsulated LiPc was unaffected by biological oxidoreductants, stable in vivo for four weeks. Oximetry, stability and biocompatibility properties of LiPc films were comparable, but both CA and PVAc films are cheaper, and easier to fabricate and handle than TAF films, making them superior.

Disulfide nitroxide biradicals, DNB, have been used for glutathione, GSH, measurements by X-band electron paramagnetic resonance, EPR, in various cells and tissues. In the present paper, the postulated potential use of DNB for EPR detection of GSH in vivo was explored. Isotopic substitution in the structure of the DNB was performed for the enhancement of its EPR spectral properties. (15)N substitution in the NO fragment of the DNB decreased the number of EPR spectral lines and resulted in an approximately two-fold increase in the signal-to-noise ratio, SNR. An additional two-fold increase in the SNR was achieved by substitution of the hydrogen atoms with deuterium resulting in narrowing the EPR lines from 1.35 G to 0.95 G. The spectral changes of DNB upon reaction with GSH and cysteine were studied in vitro in a wide range of pHs at room temperature and "body" temperature, 37 degrees C, and the corresponding bimolecular rate constants were calculated. In in vivo experiments the kinetics of the L-band EPR spectral changes after injection of DNB into ovarian xenograft tumors grown in nude mice were measured by L-band EPR spectroscopy, and analyzed in terms of the two main contributing reactions, splitting of the disulfide bond and reduction of the NO fragment. The initial exponential increase of the "monoradical" peak intensity has been used for the calculation of the GSH concentration using the value of the observed rate constant for the reaction of DNB with GSH, k(obs) (pH 7.1, 37 degrees C)=2.6 M(-1)s(-1). The concentrations of GSH in cisplatin-resistant and cisplatin-sensitive tumors were found to be 3.3 mM and 1.8 mM, respectively, in quantitative agreement with the in vitro data.

A method is presented to use continuous wave electron paramagnetic resonance imaging for rapid measurement of oxygen partial pressure in three spatial dimensions. A particulate paramagnetic probe is employed to create a sparse distribution of spins in a volume of interest. Information encoding location and spectral linewidth is collected by varying the spatial orientation and strength of an applied magnetic gradient field. Data processing exploits the spatial sparseness of spins to detect voxels with nonzero spin and to estimate the spectral linewidth for those voxels. The parsimonious representation of spin locations and linewidths permits an order of magnitude reduction in data acquisition time, compared to four-dimensional tomographic reconstruction using traditional spectral-spatial imaging. The proposed oximetry method is experimentally demonstrated for a lithium octa-n-butoxy naphthalocyanine (LiNc-BuO) probe using an L-band EPR spectrometer.

The goal of the present study was to evaluate the temporal response of particulate-based EPR oximetry probes to changes in partial pressure of oxygen (pO(2)). In order to accurately evaluate the oxygen-response time, we developed a method for rapid modulation of pO(2) in a chamber containing the probe using an oscillator-driven speaker-diaphragm setup. The apparatus was capable of producing sinusoidal changes in pO(2) at frequencies up to 300 Hz or more. The pressure-modulation setup was used to evaluate the temporal response of some of the most commonly used phthalocyanine-based particulate probes. For validation, the time-response of the probes was compared to that of a high sensitivity pressure sensor. The results revealed that some particulate probes could respond to changes in pO(2) with a temporal response of 3.3 ms (300 Hz). The observations were interpreted in the light of their crystalline packing in favor of oxygen diffusion. The results of the present study should enable the selection of probes for oximetry applications requiring high temporal resolution.

Electron paramagnetic resonance (EPR)-based oximetry is capable of quantifying oxygen content in samples. However, for a heterogeneous environment with multiple pO2 values, peak-to-peak linewidth of the composite EPR lineshape does not provide a reliable estimate of the overall pO2 in the sample. The estimate, depending on the heterogeneity, can be severely biased towards narrow components. To address this issue, we suggest a postprocessing method to recover the linewidth histogram which can be used in estimating meaningful parameters, such as the mean and median pO2 values. This information, although not as comprehensive as obtained by EPR spectral-spatial imaging, goes beyond what can be generally achieved with conventional EPR spectroscopy. Substantially shorter acquisition times, in comparison to EPR imaging, may prompt its use in clinically relevant models. For validation, simulation and EPR experiment data are presented.

Oxygenation status is a key determinant in both tumor growth and responses to therapeutic interventions. The oxygen partial pressure (pO2) was assessed using a novel pulsed electron paramagnetic resonance (EPR) spectroscopy at 750 MHz. Crystals of lithium phthalocyanine (LiPc) implanted into either squamous cell carcinoma (SCC) tumor or femoral muscle on opposing legs of mice were tested by pulsed EPR. The results showed pO2 of SCC tumor was 2.7 +/- 0.4 mmHg, while in the femoral muscle it was 6.1 +/- 0.9 mmHg. A major advantage of pulsed EPR oximetry over conventional continuous-wave (CW) EPR oximetry is the lack of influence from subject motion, while avoiding artifacts associated with modulation or power saturation. Resonators in pulsed EPR are overcoupled to minimize recovery time. This makes changes in coupling associated with object motion minimal without influencing spectral quality. Consequently, pulsed EPR oximetry enables approximately a temporal resolution of approximately one second in pO2 monitoring in conscious subjects, avoiding significant influence of anesthetics on the physiology being studied. The pO2 in SCC tumor and muscle was found to be higher without anesthesia (3.9 +/- 0.5 mmHg for tumor, 8.8 +/- 1.2 mmHg for muscle). These results support the advantage of pulsed EPR in examining pO2 in conscious animals with LiPc chronically implanted in predetermined regions., ((c) 2008 Wiley-Liss, Inc.)

We have developed a noninvasive EPR (electron paramagnetic resonance) oximetry, based on a new class of oxygen-sensing nano-particulate probe (LiNc-BuO), for simultaneous monitoring of stem-cell therapy and in situ oxygenation (partial pressure of oxygen, pO2) in a mouse model of acute myocardial infarction (AMI). AMI was induced by a permanent occlusion of left-anterior-descending (LAD) coronary artery. Skeletal myoblast (SM) cells were used for therapy. The oximetry probe was implanted in the mid-ventricular region using a needle. Tissue histological studies after 3 weeks of implantation of the probe revealed significant fibrosis, which was solely due to the needle track and not due to the probe particles. The feasibility of long-term monitoring of pO2 was established in control (non-infarct) group of hearts (> 3 months; pO2 = 15.0 +/- 1.2 mmHg,). A mixture of the probe with/without SM cells (1 x 10(5)) was implanted as a single injection in the infarcted region and the myocardial tissue pO2 at the site of cell therapy was measured for 4 weeks. The pO2 was significantly higher in infarcted hearts treated with SM cells (pO2 = 3.5 +/- 0.9 mmHg) compared to untreated hearts (pO2 = 1.6 +/- 0.7 mmHg). We have demonstrated, for the first time, the feasibility of monitoring pO2 in mouse hearts after stem cell therapy.

It is unclear whether oxygen plays a role in stem cell therapy. Hence, the determination of local oxygenation (Po(2)) in the infarct heart and at the site of transplantation may be critical to study the efficacy of cell therapy. To demonstrate this, we have developed an oxygen-sensing paramagnetic spin probes (OxySpin) to monitor oxygenation in the region of cell transplantation using electron paramagnetic resonance (EPR) spectroscopy. Skeletal myoblast (SM) cells isolated from thigh muscle biopsies of mice were labeled with OxySpin by coculturing the cells with submicron-sized (270 +/- 120 nm) particulates of the probe. Myocardial infarction was created by left coronary artery ligation in mice. Immediately after ligation, labeled SM cells were transplanted in the ischemic region of the heart. The engraftment of the transplanted cells and in situ Po(2) in the heart were monitored weekly for 4 wk. EPR measurements revealed the retention of cells in the infarcted tissue. The myocardial Po(2) at the site of SM cell therapy was significantly higher compared with the untreated group throughout the 4-wk period. Histological studies revealed differentiation and engraftment of SM cells into myotubes and increased incidence of neovascularization in the infarct region. The infarct size in the treated group was significantly decreased, whereas echocardiography showed an overall improvement in cardiac function when compared with untreated hearts. To our knowledge, this the first report detailing changes in in situ oxygenation in cell therapy. The increased myocardial Po(2) positively correlated with neoangiogenesis and cardiac function.

Methods currently available for the measurement of oxygen concentrations (oximetry) in viable tissues differ widely from each other in their methodological basis and applicability. The goal of this study was to compare two novel methods, particulate-based electron paramagnetic resonance (EPR) and OxyLite oximetry, in an experimental tumor model. EPR oximetry uses implantable paramagnetic particulates, whereas OxyLite uses fluorescent probes affixed on a fiber-optic cable. C3H mice were transplanted with radiation-induced fibrosarcoma (RIF-1) tumors in their hind limbs. Lithium phthalocyanine (LiPc) microcrystals were used as EPR probes. The pO(2) measurements were taken from random locations at a depth of approximately 3 mm within the tumor either immediately or 48 h after implantation of LiPc. Both methods revealed significant hypoxia in the tumor. However, there were striking differences between the EPR and OxyLite readings. The differences were attributed to the volume of tissue under examination and the effect of needle invasion at the site of measurement. This study recognizes the unique benefits of EPR oximetry in terms of robustness, repeatability and minimal invasiveness.

In continuous wave (CW) electron paramagnetic resonance imaging (EPRI), high quality of reconstruction in a limited acquisition time is a high priority. It has been shown for the case of 3D EPRI, that a uniform distribution of the projection data generally enhances reconstruction quality. In this work, we have suggested two data acquisition techniques for which the gradient orientations are more evenly distributed over the 4D acquisition space as compared to the existing methods. The first sampling technique is based on equal solid angle partitioning of 4D space, while the second technique is based on Fekete points estimation in 4D to generate a more uniform distribution of data. After acquisition, filtered backprojection (FBP) is applied to carry out the reconstruction in a single stage. The single-stage reconstruction improves the spatial resolution by eliminating the necessity of data interpolation in multi-stage reconstructions. For the proposed data distributions, the simulations and experimental results indicate a higher fidelity to the true object configuration. Using the uniform distribution, we expect about 50% reduction in the acquisition time over the traditional method of equal linear angle acquisition.

A novel procedure for in vivo imaging of the oxygen partial pressure (pO2) in implanted tumors is reported. The procedure uses electron paramagnetic resonance imaging (EPRI) of oxygen-sensing nanoprobes embedded in the tumor cells. Unlike existing methods of pO2 quantification, wherein the probes are physically inserted at the time of measurement, the new approach uses cells that are preinternalized (labeled) with the oxygen-sensing probes, which become permanently embedded in the developed tumor. Radiation-induced fibrosarcoma (RIF-1) cells, internalized with nanoprobes of lithium octa-n-butoxy-naphthalocyanine (LiNc-BuO), were allowed to grow as a solid tumor. In vivo imaging of the growing tumor showed a heterogeneous distribution of the spin probe, as well as oxygenation in the tumor volume. The pO2 images obtained after the tumors were exposed to a single dose of 30-Gy X-radiation showed marked redistribution as well as an overall increase in tissue oxygenation, with a maximum increase 6 hr after irradiation. However, larger tumors with a poorly perfused core showed no significant changes in oxygenation. In summary, the use of in vivo EPR technology with embedded oxygen-sensitive nanoprobes enabled noninvasive visualization of dynamic changes in the intracellular pO2 of growing and irradiated tumors., ((c) 2007 Wiley-Liss, Inc.)

Continuous wave electron paramagnetic resonance imaging for in vivo mapping of spin distribution and spectral shape requires rapid data acquisition. A spectral-spatial imaging technique is presented that provides an order of magnitude reduction in acquisition time, compared to iterative tomographic reprojection. The proposed approach assumes that spectral shapes in the sample are well-approximated by members from a parametric family of functions. A model is developed for the spectra measured with magnetic field modulation. Parameters defining the spin distribution and spectral shapes are then determined directly from the measurements using maximum a posteriori probability estimation. The approach does not suffer approximation error from limited sweep width of the main magnetic field and explicitly incorporates the variability in signal-to-noise ratio versus strength of magnetic field gradient. The processing technique is experimentally demonstrated on a one-dimensional phantom containing a nitroxide spin label with constant g-factor. Using an L-band EPR spectrometer, spectral shapes and spin distribution are accurately recovered from two projections and a spectral window which is comparable to the maximum linewidth of the sample.

The development of an injectable probe formulation, consisting of perchlorotriphenylmethyl triester radical dissolved in hexafluorobenzene, for in vivo oximetry and imaging of oxygen concentration in tissues using electron paramagnetic resonance (EPR) imaging is reported. The probe was evaluated for its oxygen sensitivity, biostability, and distribution in a radiation-induced fibrosarcoma tumor transplanted into C3H mice. Some of the favorable features of the probe are: a single narrow EPR peak (anoxic linewidth, 41 microT), high solubility in hexafluorobenzene (>12 mM), large linewidth sensitivity to molecular oxygen ( approximately 1.8 microT/mmHg), good stability in tumor tissue (half-life: 3.3 h), absence of spin-spin broadening (up to 12 mM), and lack of power saturation effects (up to 200 mW). Three-dimensional spatial and spectral-spatial (spectroscopic) EPR imaging measurements were used to visualize the distribution of the probe, as well as to obtain spatially resolved pO(2) information in the mice tumor subjected to normoxic and hyperoxic treatments. The new probe should enable unique opportunities for measurement of the oxygen concentration in tumors using EPR methods.

Electron paramagnetic resonance imaging (EPRI) provides direct detection and mapping of free radicals. The continuous wave (CW) EPRI technique, in particular, has been widely used in a variety of applications in the fields of biology and medicine due to its high sensitivity and applicability to a wide range of free radicals and paramagnetic species. However, the technique requires long image acquisition periods, and this limits its use for many in vivo applications where relatively rapid changes occur in the magnitude and distribution of spins. Therefore, there has been a great need to develop fast EPRI techniques. We report the development of a fast 3D CW EPRI technique using spiral magnetic field gradient. By spiraling the magnetic field gradient and stepping the main magnetic field, this approach acquires a 3D image in one sweep of the main magnetic field, enabling significant reduction of the imaging time. A direct one-stage 3D image reconstruction algorithm, modified for reconstruction of the EPR images from the projections acquired with the spiral magnetic field gradient, was used. We demonstrated using a home-built L-band EPR system that the spiral magnetic field gradient technique enabled a 4-7-fold accelerated acquisition of projections. This technique has great potential for in vivo studies of free radicals and their metabolism.

In continuous wave (CW) electron paramagnetic resonance imaging (EPRI), high quality of reconstructed image along with fast and reliable data acquisition is highly desirable for many biological applications. An accurate representation of uniform distribution of projection data is necessary to ensure high reconstruction quality. The current techniques for data acquisition suffer from nonuniformities or local anisotropies in the distribution of projection data and present a poor approximation of a true uniform and isotropic distribution. In this work, we have implemented a technique based on Quasi-Monte Carlo method to acquire projections with more uniform and isotropic distribution of data over a 3D acquisition space. The proposed technique exhibits improvements in the reconstruction quality in terms of both mean-square-error and visual judgment. The effectiveness of the suggested technique is demonstrated using computer simulations and 3D EPRI experiments. The technique is robust and exhibits consistent performance for different object configurations and orientations.

The broad spectrum of spin probes used for electron paramagnetic resonance imaging (EPRI) result in poor spatial resolution of the reconstructed images. Conventional deconvolution procedures can enhance the resolution to some extent but obtaining high resolution EPR images is still a challenge. In this work, we have implemented and analyzed the performance of a postacquisition deblurring technique to enhance the spatial resolution of the EPR images. The technique consists of two steps; noniterative deconvolution followed by iterative deconvolution of the acquired projections which are then projected back using filtered backprojection (FBP) to reconstruct a high resolution image. Further, we have proposed an analogous technique for iterative reconstruction algorithms such as multiplicative simultaneous iterative reconstruction technique (MSIRT) which can be a method of choice for many applications. The performance of the suggested deblurring approach is evaluated using computer simulations and EPRI experiments. Results suggest that the proposed procedure is superior to the standard FBP and standard iterative reconstruction algorithms in terms of mean-square-error (MSE), spatial resolution, and visual judgment. Although the procedure is described for 2D imaging, it can be readily extended to 3D imaging.

Electron paramagnetic resonance (EPR) oximetry is being widely used to measure the oxygen consumption of cells, mitochondria, and submitochondrial particles. However, further improvement of this technique, in terms of data analysis, is required to use it as a quantitative tool. Here, we present a new approach for quantitative analysis of cellular respiration using EPR oximetry. The course of oxygen consumption by cells in suspension has been observed to have three distinct zones: pO(2)-independent respiration at higher pO(2) ranges, pO(2)-dependent respiration at low pO(2) ranges, and a static equilibrium with no change in pO(2) at very low pO(2) values. The approach here enables one to comprehensively analyze all of the three zones together-where the progression of O(2) diffusion zones around each cell, their overlap within time, and their potential impact on the measured pO(2) data are considered. The obtained results agree with previously established methods such as high-resolution respirometry measurements. Additionally, it is also demonstrated how the diffusion limitations can depend on cell density and consumption rate. In conclusion, the new approach establishes a more accurate and meaningful model to evaluate the EPR oximetry data on cellular respiration to quantify related parameters using EPR oximetry.

Fast and reliable data acquisition is a major requirement for successful and useful biological electron paramagnetic resonance imaging (EPRI) experiments. Even a technologically advanced and professionally supervised EPRI system can exhibit instabilities initiated by perturbations such as animal motion, microphonics, and temperature changes. As a result, part of an acquired data set may become corrupted with excessive noise and distortions, which in turn may degrade the quality of the reconstructed image. In this work an automated scheme to monitor the system performance and stability over the course of an experiment is demonstrated. This method ensures that the quality of the acquired data is maintained during the experiment. For this purpose, four parameters including noise content and integration of each acquired projection are quantified and measured against those of the zero-gradient (ZG) projection, which is set as a quality benchmark. Projections with parameter values that differ substantially from the expected values are identified as damaged and consequently are reacquired. Therefore, the proposed technique not only effectively monitors the quality of acquisition, it also saves a substantial amount of acquisition time because it eliminates the necessity of repeating the entire experiment in cases in which only a small fraction of the data are corrupted., (Copyright (c) 2006 Wiley-Liss, Inc.)

Magnetic field modulation in CW electron paramagnetic resonance (EPR) is used for signal detection. However, it can also distort signal lineshape. In experiments where the linewidth information is of particular importance, small modulation amplitude is usually used to limit the lineshape distortion. The use of small modulation amplitude, however, results in low signal-to-noise ratio and therefore affects the precision of linewidth measurements. Recently, a new spectral simulation model has been developed enabling accurate fitting of modulation-broadened EPR spectra in liquids. Since the use of large modulation amplitude (over-modulation) can significantly enhance the EPR signal, the precision of linewidth measurements is therefore greatly improved. We investigated the over-modulation technique in EPR oximetry experiments using the oxygen-sensing probe lithium octa-n-butoxy-substitued naphthalocyanine (LiNc-BuO). Modulation amplitudes 2-18 times the intrinsic linewidth of the probe were applied to increase the spectral signal-to-noise ratio. The intrinsic linewidth of the probe at different oxygen concentrations was accurately extracted through curve fitting from the enhanced spectra. Thus, we demonstrated that the over-modulation model is also applicable to particulate oxygen-sensing probes such as LiNc-BuO and that the lineshape broadening induced by oxygen is separable from that induced by over-modulation. Therefore, the over-modulation technique can be used to enhance sensitivity and improve linewidth measurements for EPR oximetry with particulate oxygen-sensing probes with Lorentzian lineshape. It should be particularly useful for in vivo oxygen measurements, in which direct linewidth measurements may not be feasible due to inadequate signal-to-noise ratio.

The International Conference on Electron Paramagnetic Resonance Spectroscopy and Imaging of Biological Systems (EPR 2005) was held September 4 through 9, 2005, at Columbus, Ohio, U.S.A. Nearly 200 participants from 16 countries presented recent advances on the use of EPR technology to study biologic processes, with an emphasis on human health. For the first time, the EPR conference allied with the Wound Healing Conference, and the alliance opened an avenue for successful amalgamation of the basic biomedical and clinical aspects of wound healing with EPR technology and vice versa. This should lead to emerging applications of EPR technology in biomedical research and clinical practice.

Lithium octa-n-butoxy-naphthalocyanine (LiNc-BuO) is a stable free radical that can be detected by electron paramagnetic resonance (EPR) spectroscopy. Previously we have reported that microcrystals of LiNc-BuO exhibit a single sharp EPR peak, whose width varies linearly with the partial pressure of paramagnetic molecules such as oxygen and nitric oxide. In this report, we present the effect of nitrogen dioxide (NO2), which is also a paramagnetic molecule, on the EPR properties of LiNc-BuO. The gas-sensing property of LiNc-BuO is attributed to the open molecular framework of the crystal structure which is arranged with wide channels capable of accommodating large molecules such as NO2. The EPR linewidth of LiNc-BuO was highly sensitive to the partial pressure of NO2 in the gas mixture. The line-broadening was quick and reversible in the short-term for low concentration of NO2. However, the EPR signal intensity decreased with time of exposure, apparently due to a reaction of NO2 with LiNc-BuO crystals to give diamagnetic products. The results suggested that LiNc-BuO may be a useful probe for the determination of trace amounts of NO2 using EPR spectroscopy.

In electron paramagnetic resonance imaging (EPRI), long data acquisition time is one of the major problems limiting successful and useful biological EPRI experiments. Depending on the configuration (spatial distribution of paramagnetic species), information embedded in some objects can be characterized using a smaller number of projections, while others may require significantly larger number of projections to generate similar results. In order to optimize the acquisition process, it is therefore important to acquire a different number of projections for different objects. In this paper, a prediction scheme is demonstrated that can determine the number of projections required to achieve a preset reconstruction quality for a given object. After acquiring first few projections, corresponding partially filled k-space is analyzed. The complexity of data (to interpolate) in k-space is quantified and used to predict the number of required projections. All the projections are acquired using a mean-square difference-based adaptive acquisition technique that is also demonstrated in this work. The purpose of this non-uniform acquisition is to reduce redundancy in the acquired data which in turn decreases the number of projections required for the given object. It is also demonstrated that the performance of non-uniform acquisition is content dependant, and for certain configurations it may not be as effective as uniform acquisition in preserving signal from low intensity regions. The prediction scheme along with the non-uniform acquisition is tested using computer simulations, imaging of experimental phantoms, and in vivo imaging. Results indicate that the proposed method may save up to 50% of acquisition time. The techniques in this manuscript are described for 2D spatial imaging but can be extended to 3D imaging.

The absolute partial pressure of oxygen (pO(2)) in the mammary gland pad and femoral muscle of female mice was measured using EPR oximetry at 700 MHz. A small quantity of lithium phthalocyanine (LiPc) crystals was implanted in both mammary and femoral muscle tissue of female C3H mice. Subsequent EPR measurements were carried out 1-30 days after implantation with or without control of core body temperature. The pO(2) values in the tissue became stable 2 weeks after implantation of LiPc crystals. The pO(2) level was found to be higher in the femoral muscle than in the mammary tissue. However, the pO(2) values showed a strong dependence on the core body temperature of the mice. The pO(2) values were responsive to carbogen (95% O(2), 5% CO(2)) breathing even 44-58 days after the implantation of LiPc. The LiPc linewidth was also sensitive to changes in the blood supply even 60 days after implantation of the crystals. This study further validates the use of LiPc crystals and EPR oximetry for long-term non-invasive assessment of pO(2) levels in tissues, underscores the importance of maintaining normal body core temperature during the measurements, and demonstrates that mammary tissue functions at a lower pO(2) level than muscle in female C3H mice.