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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

A new electron paramagnetic resonance (EPR) method, rapid scan EPR (RS-EPR), is demonstrated for 2D spectral spatial imaging which is superior to the traditional continuous wave (CW) technique and opens new venues for in vivo imaging. Results are demonstrated at 250 MHz, but the technique is applicable at any frequency.

Abstract

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan electron-paramagnetic-resonance (RS-EPR), which can provide quantitative information under in vivo conditions on oxygen concentration, pH, redox status and concentration of signaling molecules (i.e., OH, NO). The RS-EPR technique has a higher sensitivity, improved spatial resolution (1 mm), and shorter acquisition time in comparison to the standard continuous wave (CW) technique. A variety of phantom configurations have been tested, with spatial resolution varying from 1 to 6 mm, and spectral width of the reporter molecules ranging from 16 µT (160 mG) to 5 mT (50 G). A cross-loop bimodal resonator decouples excitation and detection, reducing the noise, while the rapid scan effect allows more power to be input to the spin system before saturation, increasing the EPR signal. This leads to a substantially higher signal-to-noise ratio than in conventional CW EPR experiments.

Introduction

Relative to other medical imaging modalities, electron paramagnetic resonance imaging (EPRI) is uniquely able to quantitatively image physiological properties including pH1-3, pO24-7, temperature8, perfusion and viability of tissues9, microviscosity and ease of diffusion of small molecules10 and oxidative stress11. Estimation of the ease of disulfide cleavage by glutathione (GSH) in tissue and cells12,13 can report on redox status. For in vivo imaging, EPR in the frequency range between 250 MHz and 1 GHz is chosen because these frequencies provide sufficient depth of tissu....

Protocol

1. Setup of the Rapid Scan Coil Driver at 250 MHz

  1. Calculation of Rapid Scan Experimental Conditions
    Note: The most important parameter in RS-EPR is scan rate, α, which is the product of scan frequency and scan width (Equation 3). For narrow scan widths, faster scan rates are used, and for wider sweep widths, slower scan rates are used. The following instructions step through the latter case and show how to arrive at the experimental coil driver parameters of 7 mT sweep width and 6.......

Representative Results

The product of the experiment is a set of projections that are reconstructed into two-dimensional (one spectral, one spatial) images with a false color scale to represent signal amplitude. Deep blue denotes baseline where no signal is present, green is low amplitude and red is highest. Slices along the x-axis (spectral dimension) depict the EPR signal (EPR transition) on a magnetic field axis. Along the y-axis (spatial dimension), separation between signals corresponds to the physical spa.......

Discussion

Rapid-scan signals have higher frequency components than CW, and require a larger resonator bandwidth depending on linewidths, relaxation times, and the speed of the rapid-scans. The bandwidth required for a given experiment is based upon the linewidth and the scan rate of the magnetic field (Equation 2). Depending on the relaxation times of the probe under study (T2 and T2*), and the scan rate, oscillations can appear on the trailing edge of the signal. For nitroxide radicals with T2 ~50.......

Disclosures

We have nothing to disclose.

Acknowledgements

Partial support of this work by NIH grants NIBIB EB002807 and CA177744 (GRE and SSE) and P41 EB002034 to GRE, Howard J. Halpern, PI, and by the University of Denver is gratefully acknowledged. Mark Tseytlin was supported by NIH R21 EB022775, NIH K25 EB016040, NIH/NIGMS U54GM104942. The authors are grateful to Valery Khramtsov, now at the University of West Virginia, and Illirian Dhimitruka at the Ohio State University for synthesis of the pH sensitive TAM radicals, and to Gerald Rosen and Joseph Kao at the University of Maryland for synthesis of the mHCTPO, proxyl, BMPO and nitronyl radicals.

....

Materials

NameCompanyCatalog NumberComments
4-oxo-2,2,6,6-tetra(2H3)methyl-1-(3,3,5,5-2H4,1-15N)piperdinyloxyl (15N PDT)CDN Isotopes M-232798% atom 15N, 98 % atom D, Quebec Canada
4-1H-3-carbamoyl-2,2,5,5-tetra(2H3)methyl-3-pyrrolinyloxyl (15N mHCTPO)N/AN/ASynthesized at U.Maryland and described in Reference 29
3-carboxy-2,2,5,5-tetra(2H3)methyl-1-(3,4,4-2H3,1-15N)pyrrolidinyloxyl (15N Proxyl)N/AN/ASynthesized at U.Maryland and described in reference 25
4 mm Quartz EPR TubesWilmad Glass707-SQ-100M
4-oxo-2,2,6,6-tetra(2H3)methyl-1-(3,3,5,5-2H4)piperdinyloxyl (14N PDT)CDN IsotopesD-232898% atom D, Quebec Canada
pH sensitive trityl radical (aTAM4)Ohio State UniversityN/ASynthesized at Ohio State University and described in reference 26
Potassum Phosphate, MonobasicJ.T. Baker Chemicals1-3246
6 mm Quartz EPR TubesWilmad GlassQ-5M-6M-0-250/RB
8 mm Quartz EPR TubesWilmad GlassQ-7M-8M-0-250/RB
5-tert-butoxycarbonyl-5-methyl-1-pyrroline-N-oxide (BMPO) N/AN/ASynthesized at U.Maryland and described in reference 30
Hydrogen PeroxideSigma AldrichH1009 SIGMA30%
16 mm Quartz EPR tubeWilmad Glass16-7PP-11QTZ
Medium Pressure 450 W UV lampHanovia679-A36Fairfield, NJ
L-Glutathione, reducedSigma AldrichG470-5
NitronylNAN/ASynthesized at U.Maryland and described in reference 31
Sodium Hydroxide J.T. Baker Chemicals1-3146

References

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