Variable Field Proton-Electron Double-Resonance Imaging: Application to pH mapping of aqueous samples

Abstract

A new concept of Variable Field Proton-Electron Double-Resonance Imaging (VF PEDRI) is proposed. This allows for functional mapping using specifically designed paramagnetic probes (e.g. oxygen or pH mapping) with MRI high quality spatial resolution and short acquisition time. Studies performed at 200 G field MRI with phantoms show that a pH map of the sample can be extracted using only two PEDRI images acquired in 140 s at pre-selected EPR excitation fields providing pH resolution of 0.1 pH units and a spatial resolution of 1.25mm. Note that while concept of functional VF PEDRI was demonstrated using the pH probe, it can be applied for studies of other biologically relevant parameters of the medium such as redox state, concentrations of oxygen or glutathione using specifically designed EPR probes.

Figure 1

Protonation of imidazoline pH-sensitive radical, R1. Two main resonance structures are shown illustrating the favored structure with higher unpaired electron density on nitrogen atom N-1 in the nonprotonated form.

Figure 2

FC-PEDRI pulse sequence with two pre-excitation fields for functional applications.

Figure 3

FC-DNP spectra of the nitroxide R1 obtained in phosphate–citrate buffer (10 mM each) at pH 7.62 (a) and pH 4.98 (b). Sample volume was 5 ml. Frequency of EPR irradiation 563.2 MHz, 500 ms, TR 1140ms; NEX 1; Step size = 0.04 G; P =0.8 W. All other settings were as described in Materials and Methods. A dotted line is extended from low- and high-field peaks of the spectra (b) to aid the eye.

Figure 4

pH dependence of the hypefine splitting of the nitroxide R1 measured as the distance between the center- and high-field spectral lines of the DNP spectra. The solid line is a nonlinear least-square fit of the data to a conventional titration curve yielding hfs(RH⁺)=14.21 G, hfs(R)=15.03 G and pK_a=6.1.

Figure 5

Sequence of PEDRI images of the phantom sample of a pair of tubes, 12 mm diameter, containing 1 mM aqueous solutions of the R1 probe at pH 9 (left tube) and pH 2 (right tube). Images were acquired at EPR frequency 282 MHz (≈100 G for the EPR center field) with evolution field stepped in the range from 83.0 G to 84.4 G around the position of the low-field EPR component of the R1 triplet spectrum. The observed variation of the image intensity with the shift in EPR irradiation field, B^EPR (see Fig.1), illustrates the subsequent changes with the maximal image intensity of R form (left tube) and RH⁺ form (right tube) when B^EPR is equal to 83.2 G and 84 G, respectively. The PEDRI scan parameters were: TR, 1.1 s; TE, 20 ms; flip angle, 90⁰; matrix, 64×64; NEX, 1; FOV, 80 × 80 mm; acquisition time, 70 s.

Figure 6

pH dependence of the ratio of the high-field DNP signal amplitudes measured at EPR excitation field corresponding to maximal intensities of the RH⁺ and R forms of the R1 probe. DNP spectra were acquired at EPR frequency 562 MHz (≈200 G for the EPR center field), EPR excitation fields were equal to $B_{\mathit{\text{RH}}+}^{\mathit{\text{EPR}}}$ =214.16 G and $B_R^{\mathit{\text{EPR}}}$ =214.88 G.

Figure 7

A. pH phantom and its PEDRI images acquired at $B_{\mathit{\text{RH}}+}^{\mathit{\text{EPR}}}$ =214.16 G (Left) and $B_R^{\mathit{\text{EPR}}}$ =214.88 G (Right). The PEDRI scan parameters were: TR, 1.1 s; TE, 14 ms; flip angle, 90⁰; matrix, 64×64; NEX, 1; FOV, 80×80 mm, acquisition time, 71 s; NMR frequency, 856 KHz. B. VF PEDRI: proof of concept of functional imaging. pH map of phantom was calculated from two PEDRI images acquired at pre-selected EPR excitation fields as shown on panel A. Averaged values of pH are given near corresponding tube; functional resolution was determined from standard error calculated from the variations of the pH values inside of the individual tube and did not exceed 0.1 unit of pH. Spatial resolution of 1.25 mm was calculated as ratio between image field of view and matrices size (64×64).