Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

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 pH^1-3, pO₂^4-7, 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 cells^12,13 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 6.8 kHz scan frequency.
   1. Determine the resonator bandwidth (BW_Res).
      

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.......

Materials

Name	Company	Catalog Number	Comments
4-oxo-2,2,6,6-tetra(2H3)methyl-1-(3,3,5,5-2H4,1-15N)piperdinyloxyl (15N PDT)	CDN Isotopes	M-2327	98% atom 15N, 98 % atom D, Quebec Canada
4-1H-3-carbamoyl-2,2,5,5-tetra(2H3)methyl-3-pyrrolinyloxyl (15N mHCTPO)	N/A	N/A	Synthesized 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/A	N/A	Synthesized at U.Maryland and described in reference 25
4 mm Quartz EPR Tubes	Wilmad Glass	707-SQ-100M
4-oxo-2,2,6,6-tetra(2H3)methyl-1-(3,3,5,5-2H4)piperdinyloxyl (14N PDT)	CDN Isotopes	D-2328	98% atom D, Quebec Canada
pH sensitive trityl radical (aTAM4)	Ohio State University	N/A	Synthesized at Ohio State University and described in reference 26
Potassum Phosphate, Monobasic	J.T. Baker Chemicals	1-3246
6 mm Quartz EPR Tubes	Wilmad Glass	Q-5M-6M-0-250/RB
8 mm Quartz EPR Tubes	Wilmad Glass	Q-7M-8M-0-250/RB
5-tert-butoxycarbonyl-5-methyl-1-pyrroline-N-oxide (BMPO)	N/A	N/A	Synthesized at U.Maryland and described in reference 30
Hydrogen Peroxide	Sigma Aldrich	H1009 SIGMA	30%
16 mm Quartz EPR tube	Wilmad Glass	16-7PP-11QTZ
Medium Pressure 450 W UV lamp	Hanovia	679-A36	Fairfield, NJ
L-Glutathione, reduced	Sigma Aldrich	G470-5
Nitronyl	NA	N/A	Synthesized at U.Maryland and described in reference 31
Sodium Hydroxide	J.T. Baker Chemicals	1-3146