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.     

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.     

Three-Dimensional gated EPR imaging of the beating heart: Time-resolved measurements of free radical distribution during the cardiac contractile cycle

In vivo or ex vivo EPR imaging, EPRI, has been established as a powerful technique for determining the spatial distribution of free radicals and other paramagnetic species in living organs and tissues. While instrumentation capable of performing EPR imaging of free radicals in whole tissues and isolated organs has been previously reported, it was not possible to image rapidly moving organs such as the beating heart Therefore instrumentation was developed to enable the performance of gated-spectroscopy and imaging on isolated beating rat hearts at L-band. A synchronized pulsing and timing system capable of gated acquisitions of up to 256 images per cycle, with rates of up to 16 Hz was developed. The temporal and spatial accuracy of this instrumentation was verified using a specially designed beating heart-shaped isovolumic phantom with electromechanically driven sinusoidal motion at a cycle rate of 5 Hz. Gated EPR imaging was performed on a series of isolated rat hearts perfused with nitroxide spin labels. These hearts were paced at a rate of 6 Hz with either 16 or 32 gated images acquired per cardiac contractile cycle. The images enabled visualization of the time-dependent alterations in the free radical distribution and anatomical structure of the heart that occur during the cardiac cycle.     

Mapping ischemic risk region and necrosis in the isolated heart using EPR imaging.

Reperfusion of ischemic tissue is a common event in the treatment of heart attack and stroke. To study disease pathogenesis, methods are required to measure tissue perfusion and area at risk, as well as localized regions of injury. While histology can provide this information, its destructive nature precludes assessment of time course. Thus, there is a critical need for a noninvasive technique to obtain this information. To map myocardial redox state as a possible index of cellular ischemia and viability, electron paramagnetic resonance (EPR) imaging experiments were performed on isolated rat hearts before and after the onset of regional ischemia using nitroxide spin labels. With coronary artery occlusion, the EPR images clearly showed the risk region as a void of lower intensity that reversed upon reperfusion. The extent of risk region in the heart was similar in EPR imaging and histological measurements. The unique information obtained regarding the time course of changes in redox metabolism of the risk region and normal myocardium can provide important insights regarding the mechanisms of myocardial injury during and following ischemia.     

Resolution‐recovery for EPR imaging of free radical molecules in mice

A method of post‐processing to enhance the image resolution of the distribution of free radical molecules obtained with continuous‐wave electron paramagnetic resonance (CW‐EPR) imaging is reported. The low spatial resolution of EPR imaging, which has created difficulties in biomedical applications, was overcome by the method of resolution‐recovery for EPR imaging. High spatial resolution images for the distribution of free radical molecules with a very short relaxation time were obtained with this method. The method's two‐step postprocessing consists of conventional deconvolution and filtered back‐projection and a process of iterative deconvolution. The resolution‐recovery method was demonstrated with three‐dimensional (3D) imaging of stable nitroxyl radicals in mouse head. In phantom experiments with a solution of triarylmethyl (TAM) radicals, the spatial resolution was improved by a factor of 7 with the resolution‐recovery method. Magn Reson Med, 2009. © 2009 Wiley‐Liss, Inc.     

High-resolution sodium imaging of human brain at 7 T

The feasibility of high-resolution sodium magnetic resonance imaging on human brain at 7 T was demonstrated in this study. A three-dimensional anisotropic resolution data acquisition was used to address the challenge of low signal-to-noise ratio associated with high resolution. Ultrashort echo-time sequence was used for the anisotropic data acquisition. Phantoms and healthy human brains were studied on a whole-body 7-T magnetic resonance imaging scanner. Sodium images were obtained at two high nominal in-plane resolutions (1.72 and 0.86 mm) at a slice thickness of 4 mm. Signal-to-noise ratio in the brain image (cerebrospinal fluid) was measured as 14.4 and 6.8 at the two high resolutions, respectively. The actual in-plane resolution was measured as 2.9 and 1.6 mm, 69-86% larger than their nominal values. The quantification of sodium concentration on the phantom and brain images enabled better accuracy at the high nominal resolutions than at the low nominal resolution of 3.44 mm (measured resolution 5.5 mm) due to the improvement of in-plane resolution.     

Feasibility study of imaging a living murine tumor by electron paramagnetic resonance

An electron paramagnetic resonance image was measured for the first time from in vivo field gradient spectra of a living murine tumor (Cloudman S-91 melanoma in the tail of a DBA-2J mouse) using the paramagnetic nitroxide imaging agent 3-carboxamido-2,2,5,5-tetramethylpyrroline-1-oxyl injected into the tail vein. The experiments were accomplished at L-band frequency (1.55 GHz) with a single-turn flat-loop coil. A cross-sectional image was obtained perpendicular to the tail axis, which clearly distinguished features to the submillimeter resolution level.     

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.     

EPR/NMR co-imaging for anatomic registration of free-radical images.

While electron paramagnetic resonance imaging (EPRI) enables spatial mapping of free radicals in the whole body of small animals, it solely visualizes the free-radical distribution and does not typically provide anatomic visualization of the body. However, anatomic registration is often required for meaningful interpretation of the EPRI-derived free-radical images. An approach is reported for whole-body, EPRI-based, free-radical imaging along with proton MRI in mice. EPRI instrumentation with a 750-MHz narrow band microwave bridge and transverse oriented electric field reentrant resonator, with automatic coupling control and automatic tuning control capability, was used to map the spatial distribution of nitroxide free radicals in phantoms and in living mice, while low-field proton MRI at 16 MHz was used to define the anatomic structure to register the EPR images. Small capillary tubes containing an aqueous radical label were used as markers to enable image superimposition. With this coregistration technique, the EPRI free-radical images were precisely registered, enabling assignment of the location of the observed free-radical distribution within the organs of the mice. This technique enabled differentiation of the distribution and metabolism of nitroxide radicals within the major organs and body sites of living mice.     

Fast gated EPR imaging of the beating heart: Spatiotemporally resolved 3D imaging of free‐radical distribution during the cardiac cycle

In vivo or ex vivo electron paramagnetic resonance imaging (EPRI) is a powerful technique for determining the spatial distribution of free radicals and other paramagnetic species in living organs and tissues. However, applications of EPRI have been limited by long projection acquisition times and the consequent fact that rapid gated EPRI was not possible. Hence in vivo EPRI typically provided only time‐averaged information. In order to achieve direct gated EPRI, a fast EPR acquisition scheme was developed to decrease EPR projection acquisition time down to 10–20 ms, along with corresponding software and instrumentation to achieve fast gated EPRI of the isolated beating heart with submillimeter spatial resolution in as little as 2–3 min. Reconstructed images display temporal and spatial variations of the free‐radical distribution, anatomical structure, and contractile function within the rat heart during the cardiac cycle. Magn Reson Med, 2013. © 2012 Wiley Periodicals, Inc.     

In vivo proton electron double resonance imaging of the distribution and clearance of nitroxide radicals in mice.

Proton electron double resonance imaging (PEDRI) is an emerging technique that utilizes the Overhauser effect to enable in vivo and in vitro imaging of free radicals in biological systems. Nitroxide spin probes enable measurement of tissue redox state based on their reduction to diamagnetic hydroxylamines. PEDRI instrumentation at 0.02 T was applied to assess the ability to image the in vivo distribution, clearance, and metabolism of nitroxide radicals in living mice. Using phantoms of 2,2,5,5-tetramethyl-3-carboxylpyrrolidine-N-oxyl (PCA) in normal saline the dependence of the enhancement on RF power and spin probe concentration was determined. Enhancements of up to -23 were obtained in phantoms with 2 mM levels. Maximum enhancement of -7 was observed in vivo. Coronal images of nitroxide-infused mice enabled visualization of the kinetics of spin probe uptake and clearance in different organs including the great vessels, heart, lungs, kidneys, and bladder with an in-plane spatial resolution of 0.6 mm. PEDRI of living mice was also performed using 3-carbamoyl-proxyl and 2,2,6,6-tetramethyl-4-oxopiperidine-N-oxyl to compare the different rate of clearance and metabolism among different nitroxide probes. PCA, due to its intravascular compartmentalization, provided the sharpest contrast for the vascular system and highest enhancement values in the PEDRI images among the three nitroxides.     

Design and use of PET tomographs: the effect of slice spacing

With modern positron tomographs producing 14, 21, or more transaxial slices, the effects of slice spacing on quantitative reconstruction and three-dimensional displays must be evaluated. This analysis can be approached in terms of the partial volume effect, quantified by the recovery coefficient, or in terms of sampling theory leading to the concept of aliasing. The axial recovery coefficient varies as a function of the position of an object in relation to the slices, with greater variability for larger slice spacings and finer axial resolutions. The aliased image power varies in the same way. The variability in the recovery coefficient and aliasing increase when smaller objects are imaged. Tomographs should be designed with slice spacing approximately half the full-width at half-maximum axial resolution of the tomograph; finer spacing does not appear to confer significant advantages. Thus, quantification and display in positron tomography depend on slice spacing, resolution, and object size.     

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.     

MR Imaging of the heart with cine true fast imaging with steady-state precession: influence of spatial and temporal resolutions on left ventricular functional parameters

The influence of changes in spatial and temporal resolutions on functional parameters in the left ventricle (LV) were investigated with magnetic resonance (MR) imaging with a modified true fast imaging with steady-state precession, or FISP, two-dimensional sequence that provided temporal resolution of 21-90 msec and spatial resolution of 1-3 mm. MR imaging in the heart was performed in 15 healthy volunteers. A decrease in LV functional parameters was observed with reduced spatial and temporal resolutions. The influence of temporal resolution was more relevant.     

Electron paramagnetic resonance imaging of tumor hypoxia: enhanced spatial and temporal resolution for in vivo pO2 determination.

The time-domain (TD) mode of electron paramagnetic resonance (EPR) data collection offers a means of estimating the concentration of a paramagnetic probe and the oxygen-dependent linewidth (LW) to generate pO2 maps with minimal errors. A methodology for noninvasive pO2 imaging based on the application of TD-EPR using oxygen-induced LW broadening of a triarylmethyl (TAM)-based radical is presented. The decay of pixel intensities in an image is used to estimate T2*, which is inversely proportional to pO2. Factors affecting T2* in each pixel are critically analyzed to extract the contribution of dissolved oxygen to EPR line-broadening. Suitable experimental and image-processing parameters were obtained to produce pO2 maps with minimal artifacts. Image artifacts were also minimized with the use of a novel data collection strategy using multiple gradients. Results from a phantom and in vivo imaging of tumor-bearing mice validated this novel method of noninvasive oximetry. The current imaging protocols achieve a spatial resolution of approximately 1.0 mm and a temporal resolution of approximately 9 s for 2D pO2 mapping, with a reliable oxygen resolution of approximately 1 mmHg (0.12% oxygen in gas phase). This work demonstrates that in vivo oximetry can be performed with good sensitivity, accuracy, and high spatial and temporal resolution.     

Optimized single-slab three-dimensional spin-echo MR imaging of the brain

The development and optimization of spin-echo-based, single-slab, three-dimensional techniques for magnetic resonance imaging of the whole brain are described. T1-weighted and T2-weighted image sets with a volume resolution of 1 mm(3) and fluid-attenuated inversion-recovery image sets with a volume resolution of 3 mm(3) were obtained in acquisition times of less than 10 minutes per image set.     

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.     

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.     

The influence of image resolution on the positron emission tomographic measurement of caudate glucose consumption

The aim of this study was to investigate the influence of image resolution on (a) relative and absolute values of caudate glucose consumption (rCMRGIc) determined by positron emission tomography (PET), and (b) the detection of significant differences in these metabolic values between groups of subjects. For this purpose, raw data of cerebral accumulation of fluorine-18 fluorodeoxyglucose (FDG) obtained in 11 normal subjects and in nine patients with unilateral thalamic infarction were reconstructed using filtered backprojection with four different cut-off frequencies (CFs), yielding images with a transaxial resolution of 5.7, 7.1, 8.9 and 11 mm (full-width at half-maximum; FWHM). Absolute values of caudate rCMRGIc decreased significantly by more than 30% over the range of image resolutions studied. Bilateral ratios of caudate rCMRGIc were insensitive to variations in image resolution. Levels of significance assessing the differences in mean metabolic values between patients and controls were all below 0.01. They were, however, slightly better at image resolutions of 7.1 and 8.9 mm than at a resolution of 5.7 mm. These data indicate (a) that relative values of rCMRGIc are better suited to compare quantitative results from different PET cameras than are absolute values, and (b) that the CF used for the filtered back-projection exerts a small but not negligible influence on levels of significance assessing differences in metabolic values between groups of subjects.     

One- and two-dimensional EPR imaging studies on phantoms and plant specimens

The advantages of electron paramagnetic resonance (EPR) imaging at L-band frequencies are discussed. The construction and calibration of a low-field L-band EPR imaging spectrometer is described with capillary phantoms containing aqueous nitroxides as paramagnetic imaging agents. The peak separation induced by the magnetic field gradient is related to the object separation. The linewidth of each (first-derivative) line is used to calculate the dimensions of each paramagnetic object, which is the sum of an intrinsic and a gradient-induced component. By extrapolating the linewidth back to zero field gradient, one obtains the intrinsic linewidth correction factor in computing image size. The nonuniformity caused by deterioration of biological substructure was examined with plant stems where one capillary vessel had become leaky. Spin-label destruction, particularly by biological reducing agents, was compared for three species of plant stems.