Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2012 Nov 13.
Published in final edited form as: J Magn Reson. 2008 Dec 24;197(2):181–185. doi: 10.1016/j.jmr.2008.12.018

Pulsed EPR Imaging of Nitroxides in Mice

Fuminori Hyodo 1,2,#, Shingo Matsumoto 1,#, Nallathamby Devasahayam 1, Christopher Dharmaraj 1, Sankaran Subramanian 1, James B Mitchell 1, Murali C Krishna 1,*
PMCID: PMC3496157  NIHMSID: NIHMS418693  PMID: 19157932

Abstract

Nitroxides, unlike trityl radicals, have shorter T2s which until now were not detectable by time-domain Electron Paramagnetic Resonance (EPR) spectrometer at 300 MHz pulsed EPR 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, we tested the feasibility of detecting signals from nitroxide radicals. Several nitroxides and the trityl radical Oxo63 were tested. Among the nitroxides evaluated, deuterated 15N-Tempone (15N-PDT) was found to have the longest T2. The signal intensity profile as a function of concentration of these agents was evaluated and a bi-phasic behavior was observed; beyond a nitroxide concentration of 1.5 mM, signal intensity was found to decrease as a result of self-broadening. Imaging experiments were carried out with 15N-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 T2 of 15N-PDT. In vivo experiments were demonstrated with 15N-PDT in anesthetized mice where the distribution and metabolism of 15N-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.

Keywords: Nitroxide, Pulsed EPR, Redox, Single Point Imaging

INTRODUCTION

Nitroxides, also known as aminoxyls or nitroxyls, are stable free radicals that have unique antioxidant properties (1). Undergoing reversible redox transformations between the two three oxidation states of nitroxide, hydroxylamine, and the oxoammonium cation, nitroxides are able to mimic the catalytic activities of important antioxidant enzymes such as superoxide dismutase, catalase as well as participate in radical scavenging and H-atom donating reactions to provide stoichiometric antioxidant activities (2). Discovery of many protective modes of nitroxides in in vitro and in vivo models of oxidative stress expanded the use of nitroxide to therapeutic applications, including protection against ionizing radiation, as cancer prevention and treatment, control of hypertension and weight, and protection from damage resulting from ischemia/reperfusion injury etc (2,3).

Up to now, three imaging techniques, continuous wave (CW) Electron Paramagnetic Resonance (EPR) imaging, conventional Magnetic Resonance Imaging (MRI), and Overhauser MRI (OMRI) have been used to monitor nitroxides in living objects (4–13). The EPR imaging based on Continuous-Wave (CW) approach is a direct method for imaging paramagnetic species with large line widths such as nitroxides (4,5), though it takes relatively long time for image data acquisition especially for 3-D imaging. MRI provides anatomic image with superior spatial and temporal resolutions. Nitroxides have a single unpaired electron and can provide T1 contrast to water protons in conventional MRI (7,12). However, the molar relaxivity of nitroxides is ~ 0.2 mM−1s−1 compared to 4.0 mM−1s−1 for Gd3+ complexes. In terms of contrast enhancement per unit volume the lower relaxivity of nitroxides compared with Gd3+ complexes can be partially compensated by their large distribution volume due to small molecular size and cell-permeability of (7). In MRI, a fast Look-Locker T1-mapping sequence, instead of typical dynamic T1 weighted imaging, can quantify nitroxide levels in vivo (14). OMRI, also known as Proton Electron Double Resonance Imaging (PEDRI), is a hyperpolarized MRI method and couples the advantages of MRI for spatial resolution with the specificity of EPR (10,13). The high contrast enhancement in OMRI induced by dynamic nuclear polarization (DNP), where ~ 110 times enhancement is achievable for 14N-substituted nitroxides, confers superior contrast to noise ratio (11). The relative merits and limitations of these three imaging modalities for nitroxides were examined and the estimated redox status information was found to be similar (8).

EPR Imaging can be implemented using two different modes of signal detection namely the CW-EPR and the pulsed EPR (15). Major advantages of pulsed EPR, which also known as time domain EPR or Fourier transformed EPR, over the conventional CW-EPR are its rapid data acquisition and the lack of influence from subject motion during signal acquisition, while avoiding artifacts associated with modulation or RF power saturation (16). In both cases, initially, image formation was achieved by the collection of projections under static magnetic field gradients and the reconstruction from projections using filtered back-projection methods. Though the time-domain EPR approach has similarities with the signal collection in MRI, the Fourier imaging methods used in MRI by switching gradients cannot be used since the gradient settling times are longer than the T2 of many paramagnetic species. Recently, alternative Fourier imaging approach called Single Point Imaging (SPI) has been implemented in time-domain EPR using static field gradients (17,18). This approach forms images using pure phase encoding gradients and Fourier reconstruction. The feasibility of time-domain EPR imaging in in vivo applications has been demonstrated using trityl radicals which have long T2s in the order of several microseconds. Though nitroxides have been imaged using time-domain EPR at X-band frequencies in spite of their shorter T2s compared to trityl radicals they were not detectable by time-domain EPR at 300 MHz till now because of the relatively longer spectrometer recovery time, which inversely correlates with the resonance frequency. With recent improvements in our spectrometer with respect to the spectrometer recovery time and sensitivity coupled with the fact that only a few points in the FID at a constant time interval need to be recorded with respect to their phase in the SPI, at the various phase encoding gradients, it became possible that nitroxides can be detected and imaged using pulsed EPR imaging at 300 MHz operating frequency.

In the present study, we applied the SPI technique to time-domain EPR imaging of nitroxides and, for the first time, successfully obtained well-resolved, distortion-free image of nitroxide distribution in mice. This method opens the capability of intracellular oxygen imaging for the first time.

MATERIALS AND METHODS

Chemicals

15N-PDT (4-oxo-2,2,6,6-tetramethyl piperidine-d16-1-15N-oxyl), 15N-Tempol (4-hydroxyl-2,2,6,6-tetramethyl piperidine-1-15N-oxyl), and 15N-Tempamine (4-amino-2,2,6,6-tetramethyl piperidine-1-15N-oxyl) were purchased from CDN Isotopes inc (Quebec, Canada). The triarylmethyl radical probe Oxo63 was obtained from GE Healthcare (Amersham, UK). To investigate concentration dependency of time-domain EPR signal of nitroxide, various concentrations (0.1 mM ~ 5 mM) of nitroxides and Oxo63 solutions in deuterium oxide were prepared under aerobic condition. Other materials used were of analytical grade.

A four-tube phantom, each tube (4.7 mm i.d.) containing 500 μL of 0.5 mM 15N-PDT solution saturated with gas-mixtures 0, 5, 10, 21 % oxygen (nitrogen mixture) at room temperature for 15 min, was prepared to display the effect of T2* on the pulsed EPR image intensity from nitroxides.

Pulsed (time domain) EPR scanner and SPI technique

Technical details of the time-domain EPR spectrometer operating at 300 MHz, data acquisition based on the single point imaging (SPI) modality, image reconstruction were described in the earlier reports (18,19). Briefly, the SPI is a pure phase-encoding imaging technique. The free induction decay (FID) signals following the excitation pulse (80 ns, 80 W) were sampled using an analog-digital converter (200 mega samples/s). The repetition time (TR) of 5.5 μs corresponds to 180,000 excitations and acquisitions per second using 75° flip angle. For the FIDs collection, 1 mM solutions of three nitroxides and Oxo63 were measured with 100,000 averages. For 2D SPI imaging of the four-tube phantom, the FIDs were collected with a nested looping of the X, Z gradients and each time point in the FID undergoes phase modulation enabling 2D spatial encoding. Imaging parameters were as follows: field gradient = 0.8 Gauss/cm, number of gradient steps = 21 × 21, number of averages = 100,000.

Parallel coil resonators (25 mm i.d. with 25 mm long or 17 mm i.d. with 17 mm long) were used for the pulsed EPR imaging. The quality factor (Q value) of the coil has to be small in pulsed EPR in order to shorten the receiver recovery time i.e. dead time and the Q value in the range 20~30 would be optimal for mouse imaging at 300 MHz in our pulsed EPR system. Image reconstruction was accomplished by using code written in MATLAB (Mathworks, Cambridge, MA).

Pulsed EPR imaging of nitroxide in mice

Female C3H Hen MTV mice were supplied by the Frederick Cancer Research Center, Animal Production (Frederick, MD, USA). The animals were received at 6 weeks of age and housed five per cage in a climate-controlled, circadian rhythm-adjusted room, and were allowed food and water ad libitum. Body weight measured before the experiments was 22–28 g. Mice were anesthetized by isoflurane (3 % for induction and 1–2 % for keeping anesthesia) in medical air (750 ml/min) and mounted prone on a special holder. A pressure transducer (SA Instruments, Inc., NY) was placed on the mouse to monitor and maintain steady breathing rate at 60 ± 15 per min. During EPR measurements, core body temperature of the mouse was monitored using a non-magnetic rectal temperature probe (FISO, Quebec, Canada) and maintained at 36.5 ± 1 C° with flow of warm air. A 30 G needle was cannulated into the tail vein and extended using polyethylene tubing (PE-10) for administration of nitroxide and Oxo63 solutions. 15N-PDT (150 mM solution, 7.5 μL/g body weight) in phosphate buffered saline (PBS) was injected via tail vein, and then fast SPI scan was started. To confirm the position of kidneys, a bolus (10 μL/g body weight of 75 mM solution) of Oxo63, which is well known to accumulate into kidney, was intravenously injected into the same mouse after EPR signal of 15N-PDT diminished beyond the detectable level, and imaging was started immediately thereafter. SPI data was collected with a field gradient of 1.0 Gauss/cm, 21 × 21 gradient steps, and 10,000 FIDs summed per gradient setting. The data-collection time per image was 27 s. The images were processed via Fourier reconstruction of a single time point at 800 ns from the trailing edge of the pulse.

To investigate the feasibility of oxygen mapping in tumor with pulsed EPR/nitroxide, Squamous Cell Carcinoma (SCC VII) cells were subcutaneously implanted in the right hind leg 10 days before the EPR measurements. Only tumor bearing leg was placed inside a smaller resonator (17 mm i.d with 17 mm long). Immediately after 15N-PDT injection, 3D-SPI imaging was performed with following parameters: maximum field gradient sets = 0.8, 0.92, 1.1 Gauss/cm, gradient steps = 11 × 11 × 11, number of averages = 7,000, total acquisition time 2.5 min. Experiments were carried out in compliance with the Guide for the Care and Use of Laboratory Animal Resources (National Research Council, 1996) and approved by the National Cancer Institute Animal Care and Use Committee.

RESULTS AND DISCUSSION

Detecting the free induction decay signals from paramagnetic species in pulsed EPR demands instrumentation with nanosecond time resolution, compared to millisecond-second order in MRI. Typically, an excitation pulse of ~ 100 ns creates the spin coherence which lasts for only 1–2 microseconds depending on the T2 of the paramagnetic system. However, a significant portion of the FID is lost during the spectrometer recovery time which is typically 300–500 ns. While trityl radicals have FIDs which last longer than 1–2 microseconds, nitroxides with larger line widths have shorter FIDs with T2 values in the range of 300–400 ns (21). Additionally, only 1/3 of the 14N-substituted nitroxides are typically probed as a result of their spectral multiplicity (triplet spectra) compared the trityl radicals imposing additional constraints on the sensitivity. Therefore, for a given resonator and input power, the resonator recovery, which is the major determinant in the spectrometer recovery time must be minimized, which can be accomplished by reducing the Q. Reduction of resonator Q by over-coupling was found to be advantageous in terms of the achievable B1 value and short dead times. Additional Q lowering is often achieved by resistive damping. The reduction of Q-value of the parallel coil resonator with Q values in the range of 20~25 achieved by resistive damping and over-coupling, resulted in a dead time of ~ 300 ns for an input pulse of 80 ns width and 200 W power.

To examine the feasibility of detecting FIDs from nitroxides under the current spectrometer conditions, three different nitroxides were tested by time-domain EPR at 300 MHz and compared with the well known trityl radical, Oxo63 which has been used extensively in vitro and in vivo studies at this frequency range. Figure 1 shows the FIDs of three different 15N-substituted piperidine nitroxides (15N-PDT, 15N-Tempol, and 15N-Tempamine) and Oxo63 in the absence of magnetic field gradients. It can be seen that, following the dead time, the FIDs of 15N-Tempol and 15N-Tempamine lasted for an additional 500 ns before disappearing in the noise level whereas the FID of Oxo63 lasted longer than 2 μs. The deuterated 15N-PDT had a longer FID compared with other two nitroxides and its FID was detectable up to 1.5 μs. The T2* values calculated from the FIDs were 92, 94, 196, and 450 ns for 15N-Tempamine, 15N-Tempol, 15N-PDT, and Oxo63, respectively. EPR signal remaining beyond the spectrometer dead time over total EPR signal were 3.9, 4.2, 21.7, and 50.9 % for 15N-Tempamine, 15N-Tempol, 15N-PDT, and Oxo63, respectively. These signals lasted for shorter time intervals in imaging experiments where additional field gradients dephase the FIDs faster.

Fig. 1.

Fig. 1

Open in a new tab

FID signal of nitroxides by 300 MHz pulsed EPR spectroscopy. Pulsed EPR measurement of 1 mM solutions of three 15N-substituted nitroxides (15N-PDT, 15N-Tempol, and 15N-Tempamine) and Oxo63 was performed under medical air saturation. The FID of 15N-PDT lasted beyond 1.5 μs after the excitation pulse, whereas FIDs of other two nitroxides 15N-Tempol and 15N-Tempamine disappeared under noise level within 1 μs. FID of Oxo63 could be detected longer than 2 μs after the excitation pulse.

The signal intensities of the various nitroxides and the trityl Oxo63 were monitored at different concentrations. The pulsed EPR signal intensity profile of nitroxides as a function of concentration was found to be biphasic and the shortening of T2* manifested at higher concentrations (Fig. 2A) mainly due to self broadening. EPR signal intensity of nitroxides increased with concentrations, reached maximum at 1.5 mM for 15N-PDT and 2.0 mM for 15NTempol and 15N-Tempamine, and started decreasing at higher concentrations. In contrast, EPR signal intensity of Oxo63 linearly increased till the concentration of 5 mM (Fig. 2B). The linear behavior exhibited by Oxo63 in this concentration range is consistent with its larger molecular size compared to nitroxides.

Fig. 2.

Fig. 2

Open in a new tab

Concentration dependency of pulsed EPR signal intensity of nitroxides. A) Pulsed EPR signal intensity of 15N-PDT reached maximum at 1.5 mM, whereas the other two nitroxides 15N-Tempol and 15N-Tempamine provided strongest signal at 2 mM. Biphasic manner of concentration dependency of pulsed EPR signal of nitroxides presumably due to self EPR line broadening at higher concentrations, resulting in the shortening of T2*. B) Pulsed EPR signal intensity of Oxo63 increased linearly at least till 5 mM.

Based on the FID signal properties shown in Figure 1 and 2, 15N-PDT was selected for the in vivo imaging studies. It should be noted that prior studies have shown that the nitroxide 15N-PDT is capable of permeating the cell membrane and therefore can localize both in intraand extracellular locations unlike the trityl radical Oxo63, which because of the charge and substituents, is restricted to extracellular spaces. The pulsed EPR/SPI technique was applied to imaging the distribution of 15N-PDT in mice after intravenous administration and the result is shown in Figure 3A. The dosage of 15N-PDT was optimized by estimating dilution in blood to provide maximum EPR signal intensity based on the concentration dependency as shown in Figure 2 data, and delivered via tail vein and the images were obtained 20 seconds after injection. Significant distribution of 15N-PDT was observed in vivo after intravenous injection. A series of 2D images taken as a function of time show that the intensity decreased rapidly with time, a phenomenon previously noticed with this class of compounds using CW-EPR and MRI (5,7,20). After the signal from 15N-PDT has diminished beyond the detection level, another set of image data were collected after injecting the cell-impermeable Oxo63. The EPR image of Oxo63 obtained 20 s after injection was similar to that of 15N-PDT, and the accumulation of Oxo63 in the kidneys regions was observed.

Fig. 3.

Fig. 3

Open in a new tab

Pulsed EPR imaging of nitroxide in mice. A) The first pulsed EPR image of nitroxide distribution in mouse. B) Pulsed EPR image of Oxo63 injected into the same mouse after 15N-PDT signal disappeared.

Figure 4A shows the SPI image of four tube phantom containing 15N-PDT solution equilibrated with gases containing different concentrations of oxygen and therefore imparting different line widths and consequent shortening of T2*. The image intensities from the four tube phantom show that the EPR image intensity decreased along with increase in the oxygen concentration. This is explained in terms of the interaction of paramagnetic oxygen molecule to shorten the T2* of nitroxides, resulting in further signal loss within the dead time at higher oxygen concentrations. After demonstrating the capability to image the nitroxide 15N-PDT at different levels of oxygen, the SPI/15N-PDT system was applied to tumor oxygen imaging. The SCC tumor bearing leg was placed inside the smaller parallel coil resonator (Fig. 4B). FOV size was limited to the tumor bearing leg region to compress the 3D-SPI data acquisition time to be completed before the EPR signal of 15N-PDT diminished below the noise level after intravenous injection ~ 2.5 min. Figure 4C shows coronal and axial slice views selected from 3D-SPI spin density map of 15N-PDT in tumor bearing leg and corresponding oxygen images calculated from line width distribution of 15N-PDT. Significant hypoxic region (< 10 mmHg) existed in the SCC tumor bearing mouse leg, which oxygen level was similar to or slightly lower than our previous results using Oxo63 as non-membrane permeable oxygen sensitive contrast agent in the same tumor model at the same timing (18).

Fig. 4.

Fig. 4

Open in a new tab

3D-pulsed EPR imaging of tumor oxygenation using nitroxide. A) SPI image of four tube phantom containing 0.5 mM 15N-PDT solution saturated with 0, 1, 5, 21 % of oxygen gases. Pulsed EPR signal intensity decreased with increased oxygen concentrations because of T2* shortening effect of paramagnetic oxygen molecule. B) Schematic illustration of tumor-implanted mouse setting in the smaller parallel coil resonator (17 mm i.d. with 17 mm long). C) Coronal and axial slice views selected from 3D-SPI spin density map of 15N-PDT in tumor bearing leg (top row) and corresponding oxygen images (bottom row) calculated from line width distribution of 15N-PDT.

This is the first report demonstrating the capability of detecting and imaging in vivo a cell-permeable nitroxide using pulsed EPR. With the capability of extracting oxygen dependent spectral line widths, this study points to the feasibility of probing intracellular concentrations of oxygen. Additionally, since nitroxide radicals have the flexibility of being targeted to specific intracellular locations by appropriate substitutents, this study opens the possibility of using time-domain EPR to determine pO2 in specific intracellular locations.

Acknowledgement

This research was supported by the Intramural Research Program of the Center of Cancer Research, National Cancer Institute, National Institutes of Health.

References

  • 1.Mitchell JB, Samuni A, Krishna MC, DeGraff WG, Ahn MS, Samuni U, Russo A. Biologically active metal-independent superoxide dismutase mimics. Biochemistry. 1990;29(11):2802–2807. doi: 10.1021/bi00463a024. [DOI] [PubMed] [Google Scholar]
  • 2.Soule BP, Hyodo F, Matsumoto K, Simone NL, Cook JA, Krishna MC, Mitchell JB. The chemistry and biology of nitroxide compounds. Free Radic Biol Med. 2007;42(11):1632–1650. doi: 10.1016/j.freeradbiomed.2007.02.030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Metz JM, Smith D, Mick R, Lustig R, Mitchell J, Cherakuri M, Glatstein E, Hahn SM. A phase I study of topical Tempol for the prevention of alopecia induced by whole brain radiotherapy. Clin Cancer Res. 2004;10(19):6411–6417. doi: 10.1158/1078-0432.CCR-04-0658. [DOI] [PubMed] [Google Scholar]
  • 4.Kuppusamy P, Li H, Ilangovan G, Cardounel AJ, Zweier JL, Yamada K, Krishna MC, Mitchell JB. Noninvasive imaging of tumor redox status and its modification by tissue glutathione levels. Cancer Res. 2002;62(1):307–312. [PubMed] [Google Scholar]
  • 5.Sato-Akaba H, Fujii H, Hirata H. Development and testing of a CW-EPR apparatus for imaging of short-lifetime nitroxyl radicals in mouse head. J Magn Reson. 2008 doi: 10.1016/j.jmr.2008.04.035. [DOI] [PubMed] [Google Scholar]
  • 6.Matsumoto S, Nagai M, Yamada K, Hyodo F, Yasukawa K, Muraoka M, Hirata H, Ono M, Utsumi H. A composite resonator assembly suitable for EPR/NMR coregistration imaging. Conc Magn Reson part B Magn Reson Engineering. 2005;25B(1):1–11. [Google Scholar]
  • 7.Hyodo F, Matsumoto K, Matsumoto A, Mitchell JB, Krishna MC. Probing the intracellular redox status of tumors with magnetic resonance imaging and redoxsensitive contrast agents. Cancer Res. 2006;66(20):9921–9928. doi: 10.1158/0008-5472.CAN-06-0879. [DOI] [PubMed] [Google Scholar]
  • 8.Hyodo F, Murugesan R, Matsumoto K, Hyodo E, Subramanian S, Mitchell JB, Krishna MC. Monitoring redox-sensitive paramagnetic contrast agent by EPRI, OMRI and MRI. J Magn Reson. 2008;190(1):105–112. doi: 10.1016/j.jmr.2007.10.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Matsumoto K, Hyodo F, Matsumoto A, Koretsky AP, Sowers AL, Mitchell JB, Krishna MC. High-resolution mapping of tumor redox status by magnetic resonance imaging using nitroxides as redox-sensitive contrast agents. Clin Cancer Res. 2006;12(8):2455–2462. doi: 10.1158/1078-0432.CCR-05-2747. [DOI] [PubMed] [Google Scholar]
  • 10.Lurie DJ, Hutchison JMS, Bell LH, Nicholson I, Bussell DM, Mallard JR. Field-cycled proton-electron double-resonance imaging of free radicals in large aqueous samples. J Magn Reson. 1989;84:431–437. [Google Scholar]
  • 11.Matsumoto S, Yamada K, Hirata H, Yasukawa K, Hyodo F, Ichikawa K, Utsumi H. Advantageous application of a surface coil to EPR irradiation in overhauser-enhanced MRI. Magn Reson Med. 2007;57(4):806–811. doi: 10.1002/mrm.21198. [DOI] [PubMed] [Google Scholar]
  • 12.Swartz HM, Chen K, Pals M, Sentjurc M, Morse PD., 2nd Hypoxia-sensitive NMR contrast agents. Magn Reson Med. 1986;3(1):169–174. doi: 10.1002/mrm.1910030126. [DOI] [PubMed] [Google Scholar]
  • 13.Utsumi H, Yamada K, Ichikawa K, Sakai K, Kinoshita Y, Matsumoto S, Nagai M. Simultaneous molecular imaging of redox reactions monitored by Overhauser-enhanced MRI with 14N- and 15N-labeled nitroxyl radicals. Proc Natl Acad Sci U S A. 2006;103(5):1463–1468. doi: 10.1073/pnas.0510670103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Hyodo F, Chuang KH, Goloshevsky AG, Sulima A, Griffiths GL, Mitchell JB, Koretsky AP, Krishna MC. Brain redox imaging using blood-brain barrier-permeable nitroxide MRI contrast agent. J Cereb Blood Flow Metab. 2008;28(6):1165–1174. doi: 10.1038/jcbfm.2008.5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Subramanian S, Matsumoto K, Mitchell JB, Krishna MC. Radio frequency continuous-wave and time-domain EPR imaging and Overhauser-enhanced magnetic resonance imaging of small animals: instrumental developments and comparison of relative merits for functional imaging. NMR Biomed. 2004;17(5):263–294. doi: 10.1002/nbm.897. [DOI] [PubMed] [Google Scholar]
  • 16.Matsumoto S, Espey MG, Utsumi H, Devasahayam N, Matsumoto K, Matsumoto A, Hirata H, Wink DA, Kuppusamy P, Subramanian S, Mitchell JB, Krishna MC. Dynamic monitoring of localized tumor oxygenation changes using RF pulsed electron paramagnetic resonance in conscious mice. Magn Reson Med. 2008;59(3):619–625. doi: 10.1002/mrm.21500. [DOI] [PubMed] [Google Scholar]
  • 17.Subramanian S, Devasahayam N, Murugesan R, Yamada K, Cook J, Taube A, Mitchell JB, Lohman JA, Krishna MC. Single-point (constant-time) imaging in radiofrequency Fourier transform electron paramagnetic resonance. Magn Reson Med. 2002;48(2):370–379. doi: 10.1002/mrm.10199. [DOI] [PubMed] [Google Scholar]
  • 18.Matsumoto S, Hyodo F, Subramanian S, Devasahayam N, Munasinghe J, Hyodo E, Gadisetti C, Cook JA, Mitchell JB, Krishna MC. Low-field paramagnetic resonance imaging of tumor oxygenation and glycolytic activity in mice. J Clin Invest. 2008;118(5):1965–1973. doi: 10.1172/JCI34928. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Devasahayam N, Subramanian S, Murugesan R, Hyodo F, Matsumoto K, Mitchell JB, Krishna MC. Strategies for improved temporal and spectral resolution in in vivo oximetric imaging using time-domain EPR. Magn Reson Med. 2007;57(4):776–783. doi: 10.1002/mrm.21194. [DOI] [PubMed] [Google Scholar]
  • 20.Matusmoto S, Koshiishi I, Inoguchi T, Nawata H, Utsumi H. Confirmation of superoxide generation via xanthine oxidase in streptozotocin-induced diabetic mice. Free Radic Biol Med. 2003;37:767–772. doi: 10.1080/1071576031000107344. [DOI] [PubMed] [Google Scholar]
  • 21.Tseitlin M, Dhami A, Quine RW, Rinard GA, Eaton SS, Eaton GR. Electron spin T2 of a nitroxyl radical at 250 MHz measured by rapid-scan EPR. Appl Magn Reson. 2006;30:651–656. [Google Scholar]

RESOURCES