Very-low-frequency electron paramagnetic resonance (EPR) imaging of nitroxide-loaded cells.

Recent advances in electron paramagnetic resonance (EPR) imaging have made it possible to image, in real time in vivo, cells that have been labeled with nitroxide spin probes. We previously reported that cells can be loaded to high (millimolar) intracellular concentrations with (2,2,5,5-tetramethylpyrrolidin-1-oxyl-3-ylmethyl)amine-N,N-diacetic acid by incubation with the corresponding acetoxymethyl (AM) ester. Furthermore, the intracellular lifetime (t(1/e)) of this nitroxide is 114 min-sufficiently long to permit in vivo imaging studies. In the present study, at a gradient of approximately 50 mT/m, we acquire and compare EPR images of a three-tube phantom, filled with either a 200-microM solution of the nitroxide, or a suspension of cells preincubated with the nitroxide AM ester. In both cases, 3-mm resolution images can be acquired with excellent signal-to-noise ratios (SNRs). These findings indicate that cells well-loaded with nitroxide are readily imageable by EPR imaging, and that in vivo tracking studies utilizing such cells should be feasible.     

Spin echo spectroscopic electron paramagnetic resonance imaging.

The use of spin echoes to obtain spectroscopic EPR images (spectral-spatial images) at 250 MHz is described. The advantages of spin echoes-larger signals than the free induction decay, better phase characteristics for Fourier transformation, and decay shapes undistorted by instrumental dead time-are clearly shown. An advantage is gained from using a crossed loop resonator that isolates the 250-W pump power by greater than 50 dB from the observer arm preamplifiers. The echo decay rates can be used to determine the oxygen content in solutions containing 1 mM trityl concentrations. Two- and three-dimensional images of oxygen concentration are presented.     

In vivo imaging of a stable paramagnetic probe by pulsed-radiofrequency electron paramagnetic resonance spectroscopy

Imaging of free radicals by electron paramagnetic resonance (EPR) spectroscopy using time domain acquisition as in nuclear magnetic resonance (NMR) has not been attempted because of the short spin-spin relaxation times, typically under 1 μs, of most biologically relevant paramagnetic species. Recent advances in radiofrequency (RF) electronics have enabled the generation of pulses of the order of 10–50 ns. Such short pulses provide adequate spectral coverage for EPR studies at 300 MHz resonant frequency. Acquisition of free induction decays (FID) of paramagnetic species possessing inhomogenously broadened narrow lines after pulsed excitation is feasible with an appropriate digitizer/averager. This report describes the use of time-domain RF EPR spectrometry and imaging for in vivo applications. FID responses were collected from a water-soluble, narrow line width spin probe within phantom samples in solution and also when infused intravenously in an anesthetized mouse. Using static magnetic field gradients and back-projection methods of image reconstruction, two-dimensional images of the spin-probe distribution were obtained in phantom samples as well as in a mouse. The resolution in the images was better than 0.7 mm and devoid of motional artifacts in the in vivo study. Results from this study suggest a potential use for pulsed RF EPR imaging (EPRI) for three-dimensional spatial and spectral-spatial imaging applications. In particular, pulsed EPRI may find use in in vivo studies to minimize motional artifacts from cardiac and lung motion that cause significant problems in frequency-domain spectral acquisition, such as in continuous wave (cw) EPR techniques.     

Slice-selective images of free radicals in mice with modulated field gradient electron paramagnetic resonance (EPR) imaging.

Continuous wave (CW) electron paramagnetic resonance (EPR) imaging can be used to obtain slice-selective images of free radicals without measuring three-dimensional (3D) projection data. A method that incorporated a modulated magnetic field gradient (MFG) was combined with polar field gradients to select a slice in the subject noninvasively. The slice-selective in vivo EPR imaging of triarylmethyl radicals in the heads of live mice is reported. 3D surface-rendered images were successfully obtained from slice-selective images. In the experiment in mice, a slice thickness of 1.8 mm was achieved.     

A forward-subtraction procedure for removing hyperfine artifacts in electron paramagnetic resonance imaging

The potential for using electron paramagnetic resonance (EPR) imaging in biological applications has been limited by the lack of ideal single-line imaging probes. The commonly used nitroxides exhibit multiple lines, causing either hyper-fine-based limitations in the maximum obtainable image resolution or hyperfine-based artifacts in the reconstructed image. The application of a numerical method, based on forward-subtraction principles for removing hyperfine artifacts in the measured projections is reported. It is demonstrated by using computer simulations, imaging of phantoms, and imaging of rat hearts, that marked enhancement in image quality and resolution can be obtained by removing the hyper-fine-imposed limit on the gradient magnitude and performing postacquisition corrections for removing hyperfine artifacts in the image.     

High resolution electron paramagnetic resonance imaging of biological samples with a single line paramagnetic label

The application of electron paramagnetic resonance imaging (EPRI) to obtain information from biological samples has been limited by the lack of ideal single line radical labels. The commonly used nitroxides exhibit multiple lines causing either hyperfine-based limitations in the maximum obtainable image resolution or hyperfine-based artifacts in the reconstructed image. The use of a novel single-line triarylmethyl paramagnetic label that enables marked enhancement in image quality and resolution is reported. This label exhibits a single line EPR spectrum that is sharp (linewidth ～60 mG in the absence of oxygen) and relatively stable in tissues. The potential of this label in enabling high resolution EPR imaging of biological samples was demonstrated in a series of phantoms and isolated biological organs such as the rat kidney. The images demonstrate that resolutions better than 100 μm could be obtained at L-band on samples of up to 20 mm in size.     

Spatial mapping of nitric oxide generation in the ischemic heart using electron paramagnetic resonance imaging

Recently, it has been shown that rat hearts subjected to global ischemia generate nitric oxide (NO) and that a significant portion of it is generated by the reduction of nitrite under the acidic and reducing conditions that occur during myocardial ischemia [Zweier, Wang, Samouilov, Kuppusamy, Nature Med. 1, 804–809 (1995)]. In the present study it is further attempted to map the spatial distributions of the NO generation in the ischemic myocardium using L-band electron paramagnetic resonance imaging. Rat hearts were loaded with 10 mM nitrite and subjected to global no-flow ischemia, during which time a series of three-dimensional spatial electron paramagnetic resonance (EPR) images of the distribution of NO were obtained using the NO trap bis(N-methyl-D-glucamine dithiocarbamate)iron(II). The images clearly showed that NO is formed throughout the myocardium. Kinetic experiments showed that maximum NO generation and trapping occur at the midmyocardium and spread out to endocardium and epicardium of the left ventricle. The magnitude of generation in the RV myocardium is four- to fivefold lower than in the LV.     

Proton electron double resonance imaging (PEDRI) of the isolated beating rat heart.

Proton electron double resonance imaging (PEDRI) is a double resonance technique where proton MRI is performed with irradiation of a paramagnetic solute. A low-field PEDRI system was developed at 20.1 mT suitable for imaging free radicals in biological samples. With a new small dual resonator, PEDRI was applied to image nitroxide free radicals in isolated beating rat hearts. Experiments with phantoms showed maximum image enhancement factors (IEF) of 42 or 28 with TEMPONE radical concentrations of 2-3 mM at EPR irradiation powers of 12W or 6W, respectively. In the latter case, image resolution better than 0.5 mm and radical sensitivity of 5 microM was obtained. For isolated heart studies, EPR irradiation power of 6W provided optimal compromise of modest sample heating with good SNR. Only a small increase in temperature of about 1 degrees C was observed, while cardiac function remained within 10% of control values. With infusion of 3 mM TEMPONE an IEF of 15 was observed enabling 2D or 3D images to be obtained in 27 sec or 4.5 min, respectively. These images visualized the change in radical distribution within the heart during infusion and clearance. Thus, PEDRI enables rapid and high-quality imaging of free radical uptake and clearance in perfused hearts and provides a useful technique for studying cardiac radical metabolism.     

Magnetic resonance imaging (MRI) of diastematomyelia

Summary The MRI-features of diastematomyelia in a patient with unusually late onset of symptoms are reported. Direct visualization of the split cord and low conus on frontal MR-images was facilitated by three-dimensional Fourier transform (3-DFT) image acquisition.     

Noninvasive in vivo oximetric imaging by radiofrequency FT EPR.

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.     

Three-dimensional analytical magnetic resonance imaging phantom in the Fourier domain.

This work presents a basic framework for constructing a 3D analytical MRI phantom in the Fourier domain. In the image domain the phantom is modeled after the work of Kak and Roberts on a 3D version of the famous Shepp-Logan head phantom. This phantom consists of several ellipsoids of different sizes, orientations, locations, and signal intensities (or gray levels). It will be shown that the k-space signal derived from the phantom can be analytically expressed. As a consequence, it enables one to bypass the need for interpolation in the Fourier domain when testing image-reconstruction algorithms. More importantly, the proposed framework can serve as a benchmark for contrasting and comparing different image-reconstruction techniques in 3D MRI with a non-Cartesian k-space trajectory. The proposed framework can also be adapted for 3D MRI simulation studies in which the MRI parameters of interest may be introduced to the signal intensity from the ellipsoid.     

Three-dimensional whole body imaging of spin probes in mice by time-domain radiofrequency electron paramagnetic resonance.

Imaging of stable paramagnetic spin probes in phantom objects and in vivo was evaluated using a RF time domain EPR spectrometer/imager operating at 300 MHz. Projections were collected using static magnetic field gradients and images were reconstructed using filtered back-projection techniques. Results from phantom objects containing approximately 10(17) spins of stable paramagnetic probes with single narrow EPR spectra provide three-dimensional spatial images with resolution better than 2 mm. When the spin probe was administered to mice, the spin probe accumulation was temporally observed in the thoracic, abdominal, and pelvic regions. A three-dimensional image (from 144 projections) from a live mouse was collected in 5 min. Using fiducial markers, the spin probe accumulation in organs such as liver, kidney, and bladder could be observed. Differences in the oxygen status between liver and kidney were observed from the EPR images from mice administered with spin probe, by treating the time-domain responses with convolution difference approach, prior to image reconstruction. The results from these studies suggest that, with the use of stable paramagnetic spin probes and time-domain RF EPR, it is possible to perform in vivo imaging on animals and also obtain important spatially resolved physiologic information.     

Quantitative tumor oxymetric images from 4D electron paramagnetic resonance imaging (EPRI): methodology and comparison with blood oxygen level-dependent (BOLD) MRI.

This work presents a methodology for obtaining quantitative oxygen concentration images in the tumor-bearing legs of living C3H mice. The method uses high-resolution electron paramagnetic resonance imaging (EPRI). Enabling aspects of the methodology include the use of injectable, narrow, single-line triaryl methyl spin probes and an accurate model of overmodulated spectra. Both of these increase the signal-to-noise ratio (SNR), resulting in high resolution in space (1 mm)(3) and oxygen concentrations (approximately 3 torr). Thresholding at 15% the maximum spectral amplitude gives leg/tumor shapes that reproduce those in photographs. The EPRI appears to give reasonable oxygen partial pressures, showing hypoxia (approximately 0-6 torr, 0-10(3) Pa) in many of the tumor voxels. EPRI was able to detect statistically significant changes in oxygen concentrations in the tumor with administration of carbogen, although the changes were not increased uniformly. As a demonstration of the method, EPRI was compared with nearly concurrent (same anesthesia) T(2)*/blood oxygen level-dependent (BOLD) MRI. There was a good spatial correlation between EPRI and MRI. Homogeneous and heterogeneous T(2)*/BOLD MRI correlated well with the quantitative EPRI. This work demonstrates the potential for EPRI to display, at high spatial resolution, quantitative oxygen tension changes in the physiologic response to environmental changes.