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Radiation therapy is one of the main modalities to treat cancer/tumor. The response to radiation therapy, however, can be influenced by physiological and/or pathological conditions in the target tissues, especially by the low partial oxygen pressure and altered redox status in cancer/tumor tissues. Visualizing such cancer/tumor patho-physiological microenvironment would be a useful not only for planning radiotherapy but also to detect cancer/tumor in an earlier stage. Tumor hypoxia could be sensed by positron emission tomography (PET), electron paramagnetic resonance (EPR) oxygen mapping, and in vivo dynamic nuclear polarization (DNP) MRI. Tissue oxygenation could be visualized on a real-time basis by blood oxygen level dependent (BOLD) and/or tissue oxygen level dependent (TOLD) MRI signal. EPR imaging (EPRI) and/or T 1 -weighted MRI techniques can visualize tissue redox status non-invasively based on paramagnetic and diamagnetic conversions of nitroxyl radical contrast agent. 13 C-DNP MRI can visualize glycometabolism of tumor/cancer tissues. Accurate co-registration of those multimodal images could make mechanisms of drug and/or relation of resulted biological effects clear. A multimodal instrument, such as PET-MRI, may have another possibility to link multiple functions. Functional imaging techniques individually developed to date have been converged on the concept of theranostics.

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

Purpose: Spin-lattice relaxation rate (R 1 )-based time-domain EPR oximetry is reported for in vivo applications using a paramagnetic probe, a trityl-based Oxo71., Methods: The R 1 dependence of the trityl probe Oxo71 on partial oxygen pressure (pO 2 ) was assessed using single-point imaging mode of spatial encoding combined with rapid repetition, similar to T 1 -weighted MRI, for which R 1 was determined from 22 repetition times ranging from 2.1 to 40.0 μs at 300 MHz. The pO 2 maps of a phantom with 3 tubes containing 2 mM Oxo71 solutions equilibrated at 0%, 2%, and 5% oxygen were determined by R 1 and apparent spin-spin relaxation rate ( R2*) simultaneously., Results: The pO 2 maps derived from R 1 and R2* agreed with the known pO 2 levels in the tubes of Oxo71. However, the histograms of pO 2 revealed that R 1 offers better pO 2 resolution than R2* in low pO 2 regions. The SDs of pixels at 2% pO 2 (15.2 mmHg) were about 5 times lower in R 1 -based estimation than R2*-based estimation (mean ± SD: 13.9 ± 1.77 mmHg and 18.3 ± 8.70 mmHg, respectively). The in vivo pO 2 map obtained from R 1 -based assessment displayed a homogeneous profile in low pO 2 regions in tumor xenografts, consistent with previous reports on R2*-based oximetric imaging. The scan time to obtain the R 1 map can be significantly reduced using 3 repetition times ranging from 4.0 to 12.0 μs., Conclusion: Using the single-point imaging modality, R 1 -based oximetry imaging with useful spatial and oxygen resolutions for small animals was demonstrated., (© 2018 International Society for Magnetic Resonance in Medicine.)

Purpose: To develop an implantable wireless coil with parametric amplification capabilities for time-domain electron paramagnetic resonance (EPR) spectroscopy operating at 300 MHz., Methods: The wireless coil and lithium phthalocyanine (LiPc), a solid paramagnetic probe, were each embedded individually in a biocompatible polymer polydimethoxysiloxane (PDMS). EPR signals from the LiPc embedded in PDMS (LiPc/PDMS) were generated by a transmit-receive surface coil tuned to 300 MHz. Parametric amplification was configured with an external pumping coil tuned to 600 MHz and placed between the surface coil resonator and the wireless coil., Results: Phantom studies showed significant enhancement in signal to noise using the pumping coil. However, no influence of the pumping coil on the oxygen-dependent EPR spectral linewidth of LiPc/PDMS was observed, suggesting the validity of parametric amplification of EPR signals for oximetry by implantation of the encapsulated wireless coil and LiPc/PDMS in deep regions of live objects. In vivo studies demonstrate the feasibility of this approach to longitudinally monitor tissue pO 2 in vivo and also monitor acute changes in response to pharmacologic challenges. The encapsulated wireless coil and LiPc/PDMS engendered no host immune response when implanted for ∼3 weeks and were found to be well tolerated., Conclusions: This approach may find applications for monitoring tissue oxygenation to better understand the pathophysiology associated with wound healing, organ transplantation, and ischemic diseases., (© 2018 International Society for Magnetic Resonance in Medicine.)

Significance: Electron paramagnetic resonance imaging (EPRI) is capable of generating images of tissue oxygenation using exogenous paramagnetic probes such as trityl radicals or nitroxyl radicals. The spatial distribution of the paramagnetic probe can be generated using magnetic field gradients as in magnetic resonance imaging and, from its spectral features, spatial maps of oxygen can be obtained from live objects. In this review, two methods of signal acquisition and image formation/reconstruction are described. The probes used and its application to study tumor physiology and monitor treatment response with chemotherapy drugs in mouse models of human cancer are summarized. Recent Advances: By implementing phase encoding/Fourier reconstruction in EPRI in time domain mode, the frequency contribution to the spatial resolution was avoided and images with improved spatial resolution were obtained. The EPRI-generated pO 2 maps in tumor were useful to detect and evaluate the effects of various antitumor therapies on tumor physiology. Coregistration with other imaging modalities provided a better understanding of hypoxia-related alteration in physiology., Critical Issues: The high radiofrequency (RF) power of EPR irradiation and toxicity profile of radical probes are the main obstacles for clinical application. The improvement of RF low power pulse sequences may allow for clinical translation., Future Directions: Pulsed EPR oximetry can be a powerful tool to research various diseases involving hypoxia such as cancer, ischemic heart diseases, stroke, and diabetes. With appropriate paramagnetic probes, it can also be applied for various other purposes such as detecting local acid-base balance or oxidative stress. Antioxid. Redox Signal. 28, 1378-1393.

Significance: Proton-electron double-resonance imaging (PEDRI) employs electron paramagnetic resonance irradiation with low-field magnetic resonance imaging so that the electron spin polarization is transferred to nearby protons, resulting in higher signals. PEDRI provides information about free radical distribution and, indirectly, about the local microenvironment such as partial pressure of oxygen (pO 2 ), tissue permeability, redox status, and acid-base balance. Recent Advances: Local acid-base balance can be imaged by exploiting the different resonance frequency of radical probes between R and RH + forms. Redox status can also be imaged by using the loss of radical-related signal after reduction. These methods require optimized radical probes and pulse sequences., Critical Issues: High-power radio frequency irradiation is needed for optimum signal enhancement, which may be harmful to living tissue by unwanted heat deposition. Free radical probes differ depending on the purpose of PEDRI. Some probes are less effective for enhancing signal than others, which can reduce image quality. It is so far not possible to image endogenous radicals by PEDRI because low concentrations and broad line widths of the radicals lead to negligible signal enhancement., Future Directions: PEDRI has similarities with electron paramagnetic resonance imaging (EPRI) because both techniques observe the EPR signal, directly in the case of EPRI and indirectly with PEDRI. PEDRI provides information that is vital to research on homeostasis, development of diseases, or treatment responses in vivo. It is expected that the development of new EPR techniques will give insights into novel PEDRI applications and vice versa. Antioxid. Redox Signal. 28, 1345-1364.

Purpose: Pharmacokinetics of the tri[8-carboxy-2,2,6,6-tetrakis(2-hydroxymethyl)benzo[1,2-d:4,5-d']bis(1,3)dithio-4-yl]methyl radical (Oxo63) after a single bolus and/or continuous intravenous infusion was investigated in tumor-bearing C3H mice with or without body temperature control while under anesthesia., Method: The in vivo time course of Oxo63 in blood was measured using X-band electron paramagnetic resonance spectroscopy. Distribution of Oxo63 in normal muscle and tumor tissues was obtained using a surface coil resonator and a 700-MHz electron paramagnetic resonance spectrometer. The whole-body distribution of Oxo63 was obtained by 300-MHz continuous-wave electron paramagnetic resonance imaging. The high-resolution 300-MHz time-domain electron paramagnetic resonance imaging was also carried out to probe the distribution of Oxo63., Results: Urination of mice was retarded at low body temperature, causing the concentration of Oxo63 in blood to attain high levels. However, the concentration of Oxo63 in tumor tissue was lower with no control of body temperature than active body temperature control. The nonsystemized blood flow in the tumor tissues may pool Oxo63 at lower body temperature., Conclusions: Pharmacokinetics of the contrast agent were found to be significantly affected by body temperature of the experimental animal, and can influence the probe distribution and the image patterns. Magn Reson Med 79:1212-1218, 2018. © Published 2017. This article is a U.S. Government work and is in the public domain in the USA., (© Published 2017. This article is a U.S. Government work and is in the public domain in the USA.)

In vivo decay rates of a nitroxyl contrast agent were estimated by a MR redox imaging (MRRI) technique and compared with the decay rates obtained by the electron paramagnetic resonance spectroscopy (EPRS) and imaging (EPRI). MRRI is a dynamic imaging technique employing T 1 -weighted pulse sequence, which can visualise a nitroxyl-induced enhancement of signal intensity by T 1 -weighted contrast. EPR techniques can directly measure the paramagnetic nitroxyl radical. Both the squamous cell carcinoma (SCC) tumour-bearing and normal legs of a female C3H mouse were scanned by T 1 -weighted SPGR sequence at 4.7 T with the nitroxyl radical, carbamoyl-proxyl (CmP), as the contrast agent. Similarly, the time course of CmP in normal muscle and tumour tissues was obtained using a 700-MHz EPR spectrometer with a surface coil. The time course imaging of CmP was also performed by 300 MHz CW EPR imager. EPRS and EPRI gave slower decay rates of CmP compared to the MRRI. Relatively slow decay rate at peripheral region of the tumour tissues, which was found in the image obtained by MRRI, may contribute to the slower decay rates observed by EPRS and/or the EPRI measurements. To reliably determine the tissue redox status from the reduction rates of nitroxyls such as CmP, heterogenic structure in the tumour tissue must be considered. The high spatial and temporal resolution of T 1 -weighted MRI and the T 1 -enhancing capabilities of nitroxyls support the use of this method to map tissue redox status which can be a useful biomarker to guide appropriate treatments based on the tumour microenvironment.

Purpose: Electron paramagnetic resonance (EPR) imaging has evolved as a promising tool to provide non-invasive assessment of tissue oxygenation levels. Due to the extremely short T 2 relaxation time of electrons, single point imaging (SPI) is used in EPRI, limiting achievable spatial and temporal resolution. This presents a problem when attempting to measure changes in hypoxic state. In order to capture oxygen variation in hypoxic tissues and localize cycling hypoxia regions, an accelerated EPRI imaging method with minimal loss of information is needed., Methods: We present an image acceleration technique, partial Fourier compressed sensing (PFCS), that combines compressed sensing (CS) and partial Fourier reconstruction. PFCS augments the original CS equation using conjugate symmetry information for missing measurements. To further improve image quality in order to reconstruct low-resolution EPRI images, a projection onto convex sets (POCS)-based phase map and a spherical-sampling mask are used in the reconstruction process. The PFCS technique was used in phantoms and in vivo SCC7 tumor mice to evaluate image quality and accuracy in estimating O 2 concentration., Results: In both phantom and in vivo experiments, PFCS demonstrated the ability to reconstruct images more accurately with at least a 4-fold acceleration compared to traditional CS. Meanwhile, PFCS is able to better preserve the distinct spatial pattern in a phantom with a spatial resolution of 0.6mm. On phantoms containing Oxo63 solution with different oxygen concentrations, PFCS reconstructed linewidth maps that were discriminative of different O 2 concentrations. Moreover, PFCS reconstruction of partially sampled data provided a better discrimination of hypoxic and oxygenated regions in a leg tumor compared to traditional CS reconstructed images., Conclusions: EPR images with an acceleration factor of four are feasible using PFCS with reasonable assessment of tissue oxygenation. The technique can greatly enhance EPR applications and improve our understanding cycling hypoxia. Moreover this technique can be easily extended to various MRI applications., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Purpose: Electron paramagnetic resonance imaging has surfaced as a promising noninvasive imaging modality that is capable of imaging tissue oxygenation. Due to extremely short spin-spin relaxation times, electron paramagnetic resonance imaging benefits from single-point imaging and inherently suffers from limited spatial and temporal resolution, preventing localization of small hypoxic tissues and differentiation of hypoxia dynamics, making accelerated imaging a crucial issue., Methods: In this study, methods for accelerated single-point imaging were developed by combining a bilateral k-space extrapolation technique with model-based reconstruction that benefits from dense sampling in the parameter domain (measurement of the T2 (*) decay of a free induction delay). In bilateral kspace extrapolation, more k-space samples are obtained in a sparsely sampled region by bilaterally extrapolating data from temporally neighboring k-spaces. To improve the accuracy of T2 (*) estimation, a principal component analysis-based method was implemented., Results: In a computer simulation and a phantom experiment, the proposed methods showed its capability for reliable T2 (*) estimation with high acceleration (8-fold, 15-fold, and 30-fold accelerations for 61×61×61, 95×95×95, and 127×127×127 matrix, respectively)., Conclusion: By applying bilateral k-space extrapolation and model-based reconstruction, improved scan times with higher spatial resolution can be achieved in the current single-point electron paramagnetic resonance imaging modality., (© 2014 Wiley Periodicals, Inc.)

Time-domain electron paramagnetic resonance imaging is currently a useful preclinical molecular imaging modality in experimental animals such as mice and is capable of quantitatively mapping hypoxia in tumor implants. The microseconds range relaxation times (T1 and T2) of paramagnetic tracers and the large bandwidths (tens of MHz) to be excited by electron paramagnetic resonance pulses for spatial encoding makes imaging of large objects a challenging task. The possibility of using multiple array coils to permit studies on large sized object is the purpose of the present work. Toward this end, the use of planar array coils in different configurations to image larger objects than cannot be fully covered by a single resonator element is explored. Multiple circular surface coils, which are arranged in a plane or at suitable angles mimicking a volume resonator, are used in imaging a phantom and a tumor-bearing mouse leg. The image was formed by combining the images collected from the individual coils with suitable scaling. The results support such a possibility. By multiplexing or interleaving the measurements from each element of such array resonators, one can scale up the size of the subject and at the same time reduce the radiofrequency power requirements and increase the sensitivity., (Copyright © 2013 Wiley Periodicals, Inc.)

Purpose: Electron paramagnetic resonance imaging has emerged as a promising noninvasive technology to dynamically image tissue oxygenation. Owing to its extremely short spin-spin relaxation times, electron paramagnetic resonance imaging benefits from a single-point imaging scheme where the entire free induction decay signal is captured using pure phase encoding. However, direct T2 (*)/pO2 quantification is inhibited owing to constant magnitude gradients which result in time-decreasing field of view. Therefore, conventional acquisition techniques require repeated imaging experiments with differing gradient amplitudes (typically 3), which results in long acquisition time., Methods: In this study, gridding was evaluated as a method to reconstruct images with equal field of view to enable direct T2 (*)/pO2 quantification within a single imaging experiment. Additionally, an enhanced reconstruction technique that shares high spatial k-space regions throughout different phase-encoding time delays was investigated (k-space extrapolation)., Results: The combined application of gridding and k-space extrapolation enables pixelwise quantification of T2 (*) from a single acquisition with improved image quality across a wide range of phase-encoding time delays. The calculated T2 (*)/pO2 does not vary across this time range., Conclusions: By utilizing gridding and k-space extrapolation, accurate T2 (*)/pO2 quantification can be achieved within a single data set to allow enhanced temporal resolution (by a factor of 3)., (Copyright © 2013 Wiley Periodicals, Inc.)

Narrow-line spin probes derived from the trityl radical have led to the development of fast in vivo time-domain EPR imaging. Pure phase-encoding imaging modalities based on the single-point imaging scheme have demonstrated the feasibility of three-dimensional oximetric images with functional information in minutes. In this article, we explore techniques to improve the temporal resolution and circumvent the relatively short biological half-lives of trityl probes using partial k-space strategies. There are two main approaches: one involves the use of the Hermitian character of the k-space by which only part of the k-space is measured and the unmeasured part is generated using the Hermitian symmetry. This approach is limited in success by the accuracy of numerical estimate of the phase roll in the k-space that corrupts the Hermiticy. The other approach is to measure only a judicially chosen reduced region of k-space (a centrosymmetric ellipsoid region) that more or less accounts for >70% of the k-space energy. Both of these aspects were explored in Fourier transform-EPR imaging with a doubling of scan speed demonstrated by considering ellipsoid geometry of the k-space. Partial k-space strategies help improve the temporal resolution in studying fast dynamics of functional aspects in vivo with infused spin probes., (Copyright © 2012 Wiley Periodicals, Inc.)

The hypoxic nature of tumors results in treatment resistance and poor prognosis. To spare limited oxygen for more crucial pathways, hypoxic cancerous cells suppress mitochondrial oxidative phosphorylation and promote glycolysis for energy production. Thereby, inhibition of glycolysis has the potential to overcome treatment resistance of hypoxic tumors. Here, EPR imaging was used to evaluate oxygen dependent efficacy on hypoxia-sensitive drug. The small molecule 3-bromopyruvate blocks glycolysis pathway by inhibiting hypoxia inducible enzymes and enhanced cytotoxicity of 3-bromopyruvate under hypoxic conditions has been reported in vitro. However, the efficacy of 3-bromopyruvate was substantially attenuated in hypoxic tumor regions (pO2<10 mmHg) in vivo using squamous cell carcinoma (SCCVII)-bearing mouse model. Metabolic MRI studies using hyperpolarized 13C-labeled pyruvate showed that monocarboxylate transporter-1 is the major transporter for pyruvate and the analog 3-bromopyruvate in SCCVII tumor. The discrepant results between in vitro and in vivo data were attributed to biphasic oxygen dependent expression of monocarboxylate transporter-1 in vivo. Expression of monocarboxylate transporter-1 was enhanced in moderately hypoxic (8-15 mmHg) tumor regions but down regulated in severely hypoxic (<5 mmHg) tumor regions. These results emphasize the importance of noninvasive imaging biomarkers to confirm the action of hypoxia-activated drugs., (Copyright © 2012 Wiley Periodicals, Inc.)

Electron paramagnetic resonance imaging (EPRI) can be used to noninvasively and quantitatively obtain three-dimensional maps of tumor pO₂. The paramagnetic tracer triarylmethyl (TAM), a substituted trityl radical moiety, is not toxic to animals and provides narrow isotropic spectra, which is ideal for in vivo EPR imaging experiments. From the oxygen-induced spectral broadening of TAM, pO₂ maps can be derived using EPRI. The instrumentation consists of an EPRI spectrometer and 7T magnetic resonance imaging (MRI) system both operating at a common radiofrequency of 300 MHz. Anatomic images obtained by MRI can be overlaid with pO₂ maps obtained from EPRI. With imaging times of less than 3 min, it was possible to monitor the dynamics of oxygen changes in tumor and distinguish chronically hypoxic regions from acutely hypoxic regions. In this article, the principles of pO₂ imaging with EPR and some relevant examples of tumor imaging are reviewed.

MRI using hyperpolarized (13) C-labeled pyruvate is a promising tool to biochemically profile tumors and monitor their response to therapy. This technique requires injection of pyruvate into tumor-bearing animals. Pyruvate is an endogenous entity but the influence of exogenously injected bolus doses of pyruvate on tumor microenvironment is not well understood. In this study, the effect of injecting a bolus of pyruvate on tumor oxygen status was investigated. EPR oxygen imaging revealed that the partial pressure of oxygen (pO(2)) in squamous cell carcinoma implanted in mice decreased significantly 30 min after [1-(13) C]pyruvate injection, but recovered to preinjection levels after 5 h. Dynamic contrast-enhanced-MRI studies showed that, at the dose of pyruvate used, no changes in tumor perfusion were noticed. Immunohistochemical analysis of hypoxic marker pimonidazole independently verified that the squamous cell carcinoma tumor transiently became more hypoxic by pyruvate injection. Efficacy of radiotherapy was suppressed when X-irradiation was delivered during the period of pyruvate-induced transient hypoxia. These results suggest importance of taking into account the transient decrease in tumor pO(2) after pyruvate injection in hyperpolarized (13) C MRI, because tumor oxygen status is an important factor in determining outcomes of therapies., (Copyright © 2011 Wiley Periodicals, Inc.)

Oxygen maps derived from electron paramagnetic resonance spectral-spatial imaging (EPRI) are based upon the relaxivity of molecular oxygen with paramagnetic spin probes. This technique can be combined with MRI to facilitate mapping of pO(2) values in specific anatomic locations with high precision. The co-registration procedure, which matches the physical and digital dimensions of EPR and MR images, may present the pO(2) map at the higher MRI resolution, exaggerating the spatial resolution of oxygen, making it difficult to precisely distinguish hypoxic regions from normoxic regions. The latter distinction is critical in monitoring the treatment of cancer by radiation and chemotherapy, since it is well-established that hypoxic regions are three or four times more resistant to treatment compared to normoxic regions. The aim of this article is to describe pO(2) maps based on the intrinsic resolution of EPRI. A spectral parameter that affects the intrinsic spatial resolution of EPRI is the full width at half maximum (FWHM) height of the gradient-free EPR absorption line in frequency-encoded imaging. In single point imaging too, the transverse relaxation times (T(2)(∗)) limit the resolution since the signal decays by exp(-t(p)/T(2)(∗)) where the delay time after excitation pulse, t(p), is related to the resolution. Although the spin densities of two point objects may be resolved at this separation, it is inadequate to evaluate quantitative changes of pO(2) levels since the linewidths are proportionately affected by pO(2). A spatial separation of at least twice this resolution is necessary to correctly identify a change in pO(2) level. In addition, the pO(2) values are blurred by uncertainties arising from spectral dimensions. Blurring due to noise and low resolution modulates the pO(2) levels at the boundaries of hypoxic and normoxic regions resulting in higher apparent pO(2) levels in hypoxic regions. Therefore, specification of intrinsic resolution and pO(2) uncertainties are necessary to interpret digitally processed pO(2) illustrations., (Published by Elsevier Inc.)

Electron paramagnetic resonance (EPR) oximetry at 700 MHz operating frequency employing a surface coil resonator is used to assess tissue partial pressure of oxygen (pO(2)) using paramagnetic media whose linewidth and decay constant are related to oxygen concentration. Differences in extracellular and intracellular pO(2) in squamous cell carcinoma (SCC) tumor tissue were tested using several types of water-soluble paramagnetic media, which localize extracellularly or permeate through the cell membrane. The nitroxide carboxy-PROXYL (CxP) can only be distributed in blood plasma and extracellular fluids whereas the nitroxides carbamoyl-PROXYL (CmP) and TEMPOL (TPL) can permeate cell membranes and localize intracellularly. EPR signal decay constant and the linewidth of the intravenously administered nitroxides in SCC tumor tissues implanted in mouse thigh and the contralateral normal muscle of healthy mice breathing gases with different pO(2) were compared. The pO(2) in the blood can depend on the oxygen content in the breathing gas while tissue pO(2) was not directly influenced by pO(2) in the breathing gas. The decay constants of CmP and TPL in tumor tissue were significantly larger than in the normal muscles, and lower linewidths of CmP and TPL in tumor tissue was observed. The SCC tumor showed intracellular hypoxia even though the extracellular pO(2) is similar to normal tissue in the peripheral region.

Cycling hypoxia is now a well-recognized phenomenon in animal and human solid tumors. Cycling hypoxia can exist more than 100-μm distances from a microvessel, and some of these regions have been shown to exist adjacent to normal tissue. Fluctuations in pO(2) of approximately 20 mm Hg can occur with periodicities of minutes to hours and even days. These fluctuations have been attributed to changes in erythrocyte flux, perfusion, and also development of newer vascular networks. Cycling hypoxia has been shown to induce the expression of hypoxia-inducible transcription factor-1α (HIF-1α) and also confer tumor cells and tumor vascular endothelial cells with enhanced prosurvival pathways, making tumors less responsive to radiation and chemotherapy. Imaging of cycling hypoxia in tumors can provide capabilities to help plan appropriate treatment, by taking into account the magnitude and frequency of fluctuations and also their locations adjacent to normal tissue. Electron paramagnetic resonance imaging (EPRI) provides the ability to distinguish chronic and cycling hypoxic regions and has the required spatial and temporal resolutions to provide quantitative maps of tumor pO(2). EPRI can serve as a valuable tool in examining tumor pO(2) longitudinally in response to treatment and in an experimentally chosen time window to spatially map fluctuations in pO(2) noninvasively in animal models of implanted or orthotopic tumors, with a potential for human applications., (©2010 AACR.)

Tumors exhibit fluctuations in blood flow that influence oxygen concentrations and therapeutic resistance. To assist therapeutic planning and improve prognosis, noninvasive dynamic imaging of spatial and temporal variations in oxygen partial pressure (pO(2)) would be useful. Here, we illustrate the use of pulsed electron paramagnetic resonance imaging (EPRI) as a novel imaging method to directly monitor fluctuations in oxygen concentrations in mouse models. A common resonator platform for both EPRI and magnetic resonance imaging (MRI) provided pO(2) maps with anatomic guidance and microvessel density. Oxygen images acquired every 3 minutes for a total of 30 minutes in two different tumor types revealed that fluctuation patterns in pO(2) are dependent on tumor size and tumor type. The magnitude of fluctuations in pO(2) in SCCVII tumors ranged between 2- to 18-fold, whereas the fluctuations in HT29 xenografts were of lower magnitude. Alternating breathing cycles with air or carbogen (95% O(2) plus 5% CO(2)) distinguished higher and lower sensitivity regions, which responded to carbogen, corresponding to cycling hypoxia and chronic hypoxia, respectively. Immunohistochemical analysis suggests that the fluctuation in pO(2) correlated with pericyte density rather than vascular density in the tumor. This EPRI technique, combined with MRI, may offer a powerful clinical tool to noninvasively detect variable oxygenation in tumors., ((c)2010 AACR.)

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.)

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.

Nitroxides, unlike trityl radicals, have shorter T(2)s which until now were not detectable in vivo by a time-domain pulsed Electron Paramagnetic Resonance (EPR) spectrometer at 300 MHz since their phase memory times were shorter than the spectrometer recovery times. In the current version of the time-domain EPR spectrometer with improved spectrometer recovery times, the feasibility of detecting signals from nitroxide radicals was tested. Among the nitroxides evaluated, deuterated (15)N-Tempone ((15)N-PDT) was found to have the longest T(2). The signal intensity profile as a function of concentration of these agents was evaluated and a biphasic behavior was observed; beyond a nitroxide concentration of 1.5mM, signal intensity was found to decrease as a result of self-broadening. Imaging experiments were carried out with (15)N-PDT in solutions equilibrated with 0%, 5%, 10%, and 21% oxygen using the single point imaging (SPI) modality in EPR. The image intensity in these tubes was found to depend on the oxygen concentration which in turn influences the T(2) of (15)N-PDT. In vivo experiments were demonstrated with (15)N-PDT in anesthetized mice where the distribution and metabolism of (15)N-PDT could be monitored. This study, for the first time shows the capability to image a cell-permeable nitroxide in mice using pulsed EPR in the SPI modality.

Regulation of tissue redox status is important to maintain normal physiological conditions in the living body. Disruption of redox homoeostasis may lead to oxidative stress and can induce many pathological conditions such as cancer, neurological disorders and ageing. Therefore, imaging of tissue redox status could have clinical applications. Redox imaging employing magnetic resonance imaging (MRI) with nitroxides as cell-permeable redox-sensitive contrast agents has been used for non-invasive monitoring of tissue redox status in animal models. The redox imaging applications of nitroxide electron paramagnetic resonance imaging (EPRI) and MRI are reviewed here, with a focus on application of tumour redox status monitoring. While particular emphasis has been placed on differences in the redox status in tumours compared to selected normal tissues, the technique possesses the potential to have broad applications to the study of other disease states, inflammatory processes and other circumstances where oxidative stress is implicated.

A priori knowledge of spatial and temporal changes in partial pressure of oxygen (oxygenation; pO(2)) in solid tumors, a key prognostic factor in cancer treatment outcome, could greatly improve treatment planning in radiotherapy and chemotherapy. Pulsed electron paramagnetic resonance imaging (EPRI) provides quantitative 3D maps of tissue pO(2) in living objects. In this study, we implemented an EPRI set-up that could acquire pO(2) maps in almost real time for 2D and in minutes for 3D. We also designed a combined EPRI and MRI system that enabled generation of pO(2) maps with anatomic guidance. Using EPRI and an air/carbogen (95% O(2) plus 5% CO(2)) breathing cycle, we visualized perfusion-limited hypoxia in murine tumors. The relationship between tumor blood perfusion and pO(2) status was examined, and it was found that significant hypoxia existed even in regions that exhibited blood flow. In addition, high levels of lactate were identified even in normoxic tumor regions, suggesting the predominance of aerobic glycolysis in murine tumors. This report presents a rapid, noninvasive method to obtain quantitative maps of pO(2) in tumors, reported with anatomy, with precision. In addition, this method may also be useful for studying the relationship between pO(2) status and tumor-specific phenotypes such as aerobic glycolysis.

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.)

A novel continuous wave (CW), radiofrequency (RF), electron paramagnetic resonance (EPR) oximetric imaging technique is proposed, based on the influence of oxygen concentration on the RF power saturation of the EPR resonance. A linear relationship is demonstrated between the partial oxygen pressure (pO(2)) and the normalized signal intensity (I(N)), defined as, I(N) = (I(HP) - I(LP))/I(LP), where I(LP) and I(HP) refer to signal intensities at low (P(L)) and high (P(H)) RF power levels, respectively. A formula for the determination of pO(2), derived on the basis of the experimental results, reliably estimated various oxygen concentrations in a five-tube phantom. This new technique was time-efficient and also avoided the missing angle problem associated with conventional spectral-spatial CW EPR oximetric imaging. In vivo power saturation oximetric imaging in a tumor bearing mouse clearly depicted the hypoxic foci within the tumor.

EPR spectroscopy can give biologically important information, such as tissue redox status, pO2, pH, and microviscosity, based on variation of EPR spectral characteristics (i.e., intensity, linewidth, hyperfine splitting, and spectral shape of free radical probes. EPR imaging (EPRI) can obtain 1D-3D spatial distribution of such spectral components using several combinations of magnetic field gradients. Overhauser enhanced MRI (OMRI) is a double-resonance technique of electron and nuclear spins. Because the Overhauser enhancement depends on transverse relaxation rate of the electron spin, OMRI can provide pO2 information indirectly, along with a high-resolution MR image. MRI can also indirectly detect paramagnetic behaviors of free radical contrast agents. Imaging techniques and applications relating to paramagnetic species (i.e., EPRI, OMRI, and MRI) have the potential to obtain maximally 5D information (i.e., 3D spatial + 1D spectral + 1D temporal dimensions, theoretically). To obtain suitable dimensionality, several factors, such as the EPR spectral information, spatial resolution, temporal resolution, will have to be taken into account. For this review, the EPRI, OMRI, and MRI applications for the study biological systems were evaluated for researchers to apply the method of choice and the mode of measurements to specific experimental systems.

Rapid field scan on the order of T/s using high frequency sinusoidal or triangular sweep fields superimposed on the main Zeeman field, was used for direct detection of signals without low-frequency field modulation. Simultaneous application of space-encoding rotating field gradients have been employed to perform fast CW EPR imaging using direct detection that could, in principle, approach the speed of pulsed FT EPR imaging. The method takes advantage of the well-known rapid-scan strategy in CW NMR and EPR that allows arbitrarily fast field sweep and the simultaneous application of spinning gradients that allows fast spatial encoding. This leads to fast functional EPR imaging and, depending on the spin concentration, spectrometer sensitivity and detection band width, can provide improved temporal resolution that is important to interrogate dynamics of spin perfusion, pharmacokinetics, spectral spatial imaging, dynamic oxymetry, etc.

A radiofrequency (RF) time-domain electron paramagnetic resonance (EPR) instrument operating at 300, 600, and 750 MHz was used to image tumor hypoxia with high spatial and temporal resolution. A high-speed signal-averaging Peripheral Component Interconnect (PCI) board with flexibility in the input signal level and the number of digitized samples per free induction decay (FID) was incorporated into the receive arm of the spectrometer. This enabled effective and fast averaging of FIDs. Modification of the phase-encoding protocol, and replacement of the General Purpose Interface Bus (GPIB)-based handshake with a PCI-based D/A board for direct control of the gradient amplifier decreased the gradient settling and communication overhead times by nearly two orders of magnitude. Cyclically-ordered phase sequence (CYCLOPS) phase cycling was implemented to correct for pulse imperfections and cancel out unwanted constant signals. These upgrades considerably enhanced the performance of the imager in terms of image collection time, sensitivity, and temporal resolution. We demonstrated this by collecting a large number of 2D images successively and rapidly. The results show that it is feasible to achieve accurate, 2D pO(2) maps of tumor hypoxia with 1-mm(2) resolution and minimal artifacts using a set of multigradient images within an acceptable measuring time of about 3 s, and 3D maps can be obtained in less than 1 min.

Nitroxyl radicals used as tissue redox-sensitive contrast agents in electron paramagnetic resonance (EPR) and/or NMR imaging should satisfy the following two conditions: 1) the molecules disperse into tissues rapidly, and 2) paramagnetic loss occurs by simple reduction of the radical. The pharmacokinetic trends of several nitroxyl contrast agents were compared with the results obtained by bile flow monitoring (BFM) and blood circulation monitoring (BCM) methods using X-band EPR. The nitroxyl radicals (TEMPO, TEMPONE (oxo-TEMPO), and amino-TEMPO) showed additional EPR signals in the bile that were attributed to metabolites formed during transport from blood to bile through the liver. However, the highly hydrophilic CAT-1 (trimethylammonium-TEMPO), which has low membrane permeability, showed minimal concentration in the bile. Probes that have carboxyl moiety, such as carboxy-TEMPO and carboxy-PROXYL, can be transported via anion transporter into hepatic cells. The EPR signal decay profiles of the nitroxyl radicals were simulated based on the experimental data. The simulation, which we previously applied to mouse blood, was modified to simultaneously fit the experimental results of BFM and BCM obtained with rats. The simulation data showed the simplicity/complexity of the pharmacokinetic mechanisms and that carbamoyl-PROXYL and TEMPOL (hydroxy-TEMPO) are suitable contrast agents for assessing tissue redox status.

The time-domain (TD) mode of electron paramagnetic resonance (EPR) data collection offers a means of estimating the concentration of a paramagnetic probe and the oxygen-dependent linewidth (LW) to generate pO2 maps with minimal errors. A methodology for noninvasive pO2 imaging based on the application of TD-EPR using oxygen-induced LW broadening of a triarylmethyl (TAM)-based radical is presented. The decay of pixel intensities in an image is used to estimate T2*, which is inversely proportional to pO2. Factors affecting T2* in each pixel are critically analyzed to extract the contribution of dissolved oxygen to EPR line-broadening. Suitable experimental and image-processing parameters were obtained to produce pO2 maps with minimal artifacts. Image artifacts were also minimized with the use of a novel data collection strategy using multiple gradients. Results from a phantom and in vivo imaging of tumor-bearing mice validated this novel method of noninvasive oximetry. The current imaging protocols achieve a spatial resolution of approximately 1.0 mm and a temporal resolution of approximately 9 s for 2D pO2 mapping, with a reliable oxygen resolution of approximately 1 mmHg (0.12% oxygen in gas phase). This work demonstrates that in vivo oximetry can be performed with good sensitivity, accuracy, and high spatial and temporal resolution.

The integration of modern data acquisition and digital signal processing (DSP) technologies with Fourier transform electron paramagnetic resonance (FT-EPR) imaging at radiofrequencies (RF) is described. The FT-EPR system operates at a Larmor frequency (L(f)) of 300MHz to facilitate in vivo studies. This relatively low frequency L(f), in conjunction with our approximately 10MHz signal bandwidth, enables the use of direct free induction decay time-locked subsampling (TLSS). This particular technique provides advantages by eliminating the traditional analog intermediate frequency downconversion stage along with the corresponding noise sources. TLSS also results in manageable sample rates that facilitate the design of DSP-based data acquisition and image processing platforms. More specifically, we utilize a high-speed field programmable gate array (FPGA) and a DSP processor to perform advanced real-time signal and image processing. The migration to a DSP-based configuration offers the benefits of improved EPR system performance, as well as increased adaptability to various EPR system configurations (i.e., software configurable systems instead of hardware reconfigurations). The required modifications to the FT-EPR system design are described, with focus on the addition of DSP technologies including the application-specific hardware, software, and firmware developed for the FPGA and DSP processor. The first results of using real-time DSP technologies in conjunction with direct detection bandpass sampling to implement EPR imaging at RF frequencies are presented.

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.

A novel approach to measure the time course of paramagnetic spin probe concentration in the circulating blood of a living mouse using X-band (9.4 GHz) electron paramagnetic resonance spectrometer is described. Using this technique, the pharmacokinetics of several nitroxyl spin probes was examined. The decay profiles were also independently simulated using pharmacokinetic properties as well as redox-mediated factors responsible in converting the nitroxyl radicals to the corresponding hydroxylamines. Finally, suitability of nitroxyl radicals as the probes of in vivo redox status and for radioprotection was described. The studies indicate that the six-member piperidine nitroxyls are suitable for estimating redox status in the circulation, whereas the five-member pyrrolidine nitroxyl radicals are suited for tissue redox status determination. For selective protection against radiation of normal tissues rather than cancer/tumor, efficient reoxidation of the hydroxylamine in normal tissue is preferable. Simulation results showed that for carbamoyl-PROXYL, only administration of the radical form might give radioprotection and not the hydroxylamine. However, the hydroxylamine form of TEMPOL, i.e., TEMPOL-H, may give similar radioprotection as the radical form due to efficient reoxidation in vivo.

Electron paramagnetic resonance (EPR) imaging in the continuous wave (CW) and time-domain modes, as well as Overhauser-enhanced magnetic resonance imaging in vivo is described. The review is based mainly on the CW and time-domain EPR instrumentation at 300 MHz developed in our laboratory, and the relative merits of these methods for functional in vivo imaging of small animals to assess hypoxia and tissue redox status are described. Overhauser imaging of small animals at magnetic fields in the range 10-15 mT that is being carried out in our laboratory for tumor imaging and the evaluation of tumor hypoxia based on quantitative evaluation of Overhauser enhancement is also described. Alternate approaches to spectral-spatial imaging using the transverse decay constants to infer in situ line widths and hence in vivo pO2 using CW and time-domain EPR imaging are also discussed. The nature of the spin probes used, the quality of the images obtained in all the three methods, the achievable resolution, limitations and possible future directions in small animal functional imaging with these modalities are summarized., (Copyright 2004 John Wiley & Sons, Ltd.)

A Windows based application system for data collection, Fourier reconstruction and analysis of pure phase encoded constant-time radio frequency electron paramagnetic resonance (EPR) images, is described. The graphical user interface (GUI) of the system was written in MATLAB version 5.0, using its built-in GUI utilities. Design considerations of the application system included speed, flexibility and user-friendly data display and analysis. To maximize the speed of image data collection, MATLAB's built-in C interface system, MEX was not used for data collection. Instead, MATLAB programs call the C programs from the DOS prompt directly, based on the data collection parameters entered through the GUI. Computational procedures included various digital signal-processing steps such as filtering, interpolation etc. for the Fourier reconstruction of 2D, and 3D EPR images from the pure phase encoded data. Examples of 2D images illustrating the performance of the system are presented. Although the application system has been developed for the specific purpose of EPR imaging, it can easily be adapted to other areas such as magnetic resonance microscopy as well.

In this study we report the application of continuous-wave (CW) electron paramagnetic resonance (EPR) constant-time spectral spatial imaging (CTSSI) for in vivo oxymetry. 2D and 3D SSI studies of a phantom and live mice were carried out using projection reconstruction (PR) and constant-time (CT) modalities using a CW-EPR spectrometer/imager operating at 300 MHz frequency. Distortion of line shape, which is inherent in the PR method, was minimized by the CTSSI modality. It was also found that CTSSI offers improved noise reduction, restores a smoother line shape, and gives high convergence of estimated values. Spatial resolution was also improved by CTSSI, although fundamental spectral line-width broadening was observed. Although additional corrections are required for accurate estimations of spectral line width, CTSSI was able to demonstrate distinct differences in oxygen tension between a tumor and the normal legs of a C3H mouse. The PR method, on the other hand, was unable to make such a distinction unequivocally with the triarylmethyl spin probes. CTSSI promises to be a more suitable method for quantitative in vivo oxymetric studies using radiofrequency EPR imaging (EPRI).

The application of correlation spectroscopy employing stochastic excitation and the Hadamard transform to time-domain Fourier transform electron paramagnetic resonance (FT-EPR) spectroscopy in the radiofrequency (RF) band is described. An existing, time-domain FT-EPR spectrometer system with a Larmor frequency (L(f)) of 300 MHz was used to develop this technique by incorporating a pseudo-random pulse sequence generator to output the maximum length binary sequence (MLBS, 10- and 11-bit). Software developed to control the EPR system setup, acquire the signals, and post process the data, is outlined. The software incorporates the Hadamard transform algorithm to perform the required cross-correlation of the acquired signal and the MLBS after stochastic excitation. To accommodate the EPR signals, bandwidth extension was accomplished by sampling at a rate many times faster than the RF pulse repetition rate, and subsequent digital signal processing of the data. The results of these experiments showed that there was a decrease in the total acquisition time, and an improved free induction decay (FID) signal-to-noise (S/N) ratio compared to the conventional coherent averaging approach. These techniques have the potential to reduce the RF pulse power to the levels used in continuous wave (CW) EPR while retaining the advantage of time-domain EPR methods. These methods have the potential to facilitate the progression to in vivo FT-EPR imaging of larger volumes.

This study describes the use of the single-point imaging (SPI) modality, also known as constant-time imaging (CTI), in radiofrequency (RF) Fourier transform (FT) electron paramagnetic resonance (EPR). The SPI technique, commonly used for high-resolution solid-state nuclear magnetic resonance (NMR) imaging, has been successfully applied to 2D and 3D RF-FT-EPR imaging of phantoms containing narrow-line EPR spin probes. The SPI scheme is essentially a phase-encoding technique that operates by acquiring a single data point in the free induction decay (FID) after a fixed delay (phase-encoding time), following the pulsed RF excitation, in the presence of static magnetic field gradients. Since the phase-encoding time remains constant for a given image data set, the spectral information is automatically deconvolved, providing well-resolved pure spatial images. Therefore, images obtained using SPI are artifact-free and the resolution is not significantly limited by the line width, compared to the images obtained using the conventional filtered back-projection (FBP) scheme, suggesting that the SPI modality may have advantages for EPR imaging of large objects. In this work the advantages and limitations of SPI as compared to FBP are investigated by imaging suitable phantom objects. Although SPI takes longer to perform than the FBP method, optimization of the data collection scheme may increase the temporal resolution, rendering this technique suitable for in vivo studies. Spectral information can also be extracted from a series of SPI images that are generated as a function of the delay from the excitation pulse., (Published 2002 Wiley-Liss, Inc.)

A novel method, called relaxo-oximetry, for rapid spatially resolved in vivo measurements of oxygen concentration using time-domain radiofrequency (RF) electron paramagnetic resonance (EPR) is described. Time-domain data from triaryl methyl (TAM)-based single-electron contrast agents were processed by systematic deletion of the initial time points to arrive at T2*-weighted discrimination of signal amplitudes. In experiments involving phantoms, the line widths [ approximately (T2*)(-1)] increased as a function of oxygen, and the slope (line width/pO(2)) was the same for both absorption- and magnitude-mode line shapes. Line widths derived from T2* weighting and the computed pO(2) values agreed favorably with the measured ones from phantoms of known oxygen tension. In vivo relaxo-oximetry was performed on C3H mice, and it was found that the liver was more hypoxic than the kidneys. For tumors, 2D oxygen maps were generated while the animal breathed room air or Carbogen(R) (95% O(2)/5% CO(2)). Carbogen(R) enhanced oxygen concentration within the tumor, and the pO(2) histograms showed considerable heterogeneity. Clark electrode oxygen measurements on organs and tumors were in good agreement with tissue oxygen measurements done by relaxo-oximetry. Thus, from a single spatial image data set, pO(2) measurements can be done noninvasively by relaxo-oximetry, and 3D imaging can be performed in less than 3 min., (Copyright 2002 Wiley-Liss, Inc.)

The performance of two electron paramagnetic resonance (EPR) spectrometers/imagers, one configured in pulsed mode and the other in continuous wave (CW) mode, at an operating frequency of 300 MHz is compared. Using the same resonator (except for altered Q-factors), identical samples and filling factors in the two techniques have been evaluated for their potentials and limitations for in vivo spectroscopic and imaging applications. The assessment is based on metrics such as sensitivity, spatial and temporal resolution, field of view, image artifacts, viable spin probes, and subjects of study. The spectrometer dead time limits the pulsed technique to samples with long phase memories (>275 ns). Nevertheless, for viable narrow-line spin probes, the pulsed technique offers better sensitivity and temporal resolution. The CW technique, on the other hand, does not restrict the choice at spin probes. In addition, the phase-sensitive narrow-band detection of the CW technique gives artifact-free images even for large objects. Selected examples illustrating the performance of the CW and pulsed techniques are presented to put the capabilities of the two techniques in perspective., ((C) 2002 Elsevier Science (USA).)