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.     

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.     

Carbon blacks as EPR sensors for localized measurements of tissue oxygenation.

New electron paramagnetic resonance (EPR) oximetry probes were identified in the class of carbon black materials. These compounds exhibit very high oxygen sensitivity and favorable EPR characteristics for biological applications. At low pO(2), the linewidth is particularly sensitive to changes in oxygen tension (sensitivity of 750 mG/mmHg). The application of the probes for oximetry was demonstrated in vivo: the pO(2) was measured in muscle in which the blood flow was temporarily restricted as well as in tumor-bearing mice during a carbogen breathing challenge. The responsiveness to pO(2) was stable in muscle for at least 3 months. No toxicity was observed using these materials in cellular experiments and in histological studies performed 2, 7, and 28 days after implantation. In view of their EPR characteristics (high sensitivity) as well as the well-characterized production procedure that make them available on a large scale, these probes can be considered as very promising tools for future developments in EPR oximetry.     

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.     

India ink: A potential clinically applicable EPR oximetry probe

Using a material that already is in widespread use in humans, India ink, the first EPR measurements in a human have been made, using the India ink in a pre-existing tattoo. The EPR spectra of India ink are very sensitive to the partial pressure of oxygen (PO₂), thereby making it feasible to use this approach to measure pO₂ in tissues in patients. This potentially provides a means to measure this parameter directly with a sensitivity, accuracy, and repeatability that have not been available previously, and thereby to be able to individualize and guide treatment of diseases such as cancer and peripheral vascular insufficiency.     

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.     

Mapping of the B1 field distribution of a surface coil resonator using EPR imaging.

Surface coil resonators have been widely used to perform topical EPR spectroscopy. They are usually positioned adjacent to or implanted within the body. For EPR applications these resonators have a number of important advantages over other resonator designs due to their ease of sample accessibility, mechanical fabrication, implementation of electronic tuning and coupling functions, and low susceptibility to sample motions. However, a disadvantage is their B(1) field inhomogeneity, which limits their usefulness for 3D imaging applications. We show that this problem can be addressed by mapping and correcting the B(1) field distribution. We report the use of EPR imaging (EPRI) to map the B(1) distribution of a surface coil resonator. We show that EPRI provides a fast, accurate, and reliable technique to evaluate the B(1) distribution. 3D EPRI was performed on phantoms, prepared using three different saline concentrations, to obtain the B(1) distribution. The information obtained from the phantoms was used to correct the images of living animals. With the use of this B(1) correction technique, surface coil resonators can be applied to perform 3D mapping of the distribution of free radicals in biological samples and living systems.     

In vivo Oximetry Using EPR and India Ink

Recent advances in electron paramagnetic resonance (EPR) oximetry have established the use of the particulate materials fusinite and lithium phthalocyanine (LiPc) as probes for measurement of oxygen tensions in tissues in vivo with a sensitivity and accuracy that is relevant for studying pathophysiological processes. India ink, another potentially very useful new probe for EPR oximetry, shares the critical properties of fusinite and LiPc and has the additional advantage of already having been widely used clinically with no apparent toxicity. The line width is particularly sensitive to changes in oxygen tension of less than 30 mmHg; in this range the line broadening/unit of pO2 is much greater than that of fusinite and LiPc. Over the range of biological conditions that can occur in vivo, the response of the EPR spectrum of India ink to pO₂ is independent of pH, oxidants, reductants, and the nature of the medium. In this paper we describe the relevant properties of India ink and its use to measure pO₂ in vivo in experimental animals and a human subject.     

Application of continuous-wave EPR spectral-spatial image reconstruction techniques for in vivo oxymetry: comparison of projection reconstruction and constant-time modalities.

In this study we report the application of continuous-wave (CW) electron paramagnetic resonance (EPR) constant-time spectral spatial imaging (CTSSI) for in vivo oxymetry. 2D and 3D SSI studies of a phantom and live mice were carried out using projection reconstruction (PR) and constant-time (CT) modalities using a CW-EPR spectrometer/imager operating at 300 MHz frequency. Distortion of line shape, which is inherent in the PR method, was minimized by the CTSSI modality. It was also found that CTSSI offers improved noise reduction, restores a smoother line shape, and gives high convergence of estimated values. Spatial resolution was also improved by CTSSI, although fundamental spectral line-width broadening was observed. Although additional corrections are required for accurate estimations of spectral line width, CTSSI was able to demonstrate distinct differences in oxygen tension between a tumor and the normal legs of a C3H mouse. The PR method, on the other hand, was unable to make such a distinction unequivocally with the triarylmethyl spin probes. CTSSI promises to be a more suitable method for quantitative in vivo oxymetric studies using radiofrequency EPR imaging (EPRI).     

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.     

Proton NMR observation of glycogen in vivo

Our previous solution studies of the proton relaxation properties of glycogen H1 have shown significant dipolar cross–relaxation with intra-ring H2 and inter-ring H4′ protons characterized by a correlation time τ_c= 2.7 × 10^?9 s. This leads to a significant negative Nuclear Overhauser Enhancement (NOE) of glycogen H1 following either transient or steady state perturbations of the longitudinal magnetization of dipolar coupled protons, especially H2 and H4′. Here we use the NOE to edit selectively the H1 resonance of glycogen in the rat liver in vivo using a surface coil probe. The approach shows the possibility of measuring glycogen in vivo with high sensitivity using ¹H NMR.     

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.     

Pharmacokinetics of a triarylmethyl-type paramagnetic spin probe used in EPR oximetry.

The paramagnetic spin probe Oxo63 is used in oximetric imaging studies based on electron paramagnetic resonance (EPR) methods by monitoring the oxygen-dependent linewidth while minimizing the contributions from self-broadening seen at high probe concentrations. Therefore, it is necessary to determine a suitable dose of Oxo63 for EPR-based oxygen mapping where the self-broadening effects are minimized while signal intensity adequate for imaging can be realized. A constant tissue concentration of spin probe would be useful to image a subject and assess changes in pO2 over time; accumulation or elimination of the compound in specific anatomical regions could translate to and be mistaken for changes in local pO2, especially in OMRI-based oximetry. The in vivo pharmacokinetics of the spin probe, Oxo63, after bolus and/or continuous intravenous infusion was investigated in mice using a novel approach with X-band EPR spectroscopy. The results show that the half-life in blood was 17-21 min and the clearance by excretion was 0.033-0.040 min(-1). Continuous infusion following a bolus injection of the probe was found to be effective to obtain stable plasma concentration as well as image intensity to permit reliable pO2 estimates.     

Development of a PEDRI free-radical imager using a 0.38 T clinical MRI system.

Proton electron double resonance imaging (PEDRI) uses the Overhauser effect to image the distribution of free-radicals in biological samples and animals. Standard MRI hardware and software is used, with the addition of hardware to irradiate the free-radical-of-interest's EPR resonance. For in vivo applications it must be implemented at a sufficiently low magnetic field to result in an EPR irradiation frequency that will penetrate the sample but will not cause excessive nonresonant power deposition therein. Many clinical MRI systems use resistive magnets that are capable of operating at 10-20 mT, and which could thus be used as PEDRI imagers with the addition of a small amount of extra hardware. This article describes the conversion of a 0.38 T whole-body MRI system for operation as a 20.1 mT small-animal PEDRI imager. The magnet power supply control electronics required a small modification to operate at the lower field strength, but no permanent hardware changes to the MRI console were necessary, and no software modification was required. Frequency down- and up-conversion was used on the NMR RF system, together with a new NMR/EPR dual-resonance RF coil assembly. The system was tested on phantoms containing free-radical solution, and was also used to image the distribution of a free-radical contrast agent injected intravenously into anesthetized mice.     

Noninvasive measurement of the pH inside the gut by using pH-sensitive nitroxides. An in vivo EPR study

The use of pH-sensitive probes permits the measurement of the proton activity in biological systems by EPR spectroscopy. To illustrate the potential of this technique for in vivo purposes, the authors took advantage of the oral administration of nitroxides to monitor the pH value inside the stomach of mice after administration of different antacidics. The results indicate that EPR can be a valuable tool to characterize the pH in vivo in a continuous and noninvasive way.     

T*1e and T*2e maps derived in vivo from the rat using longitudinally detected electron spin resonance phase imaging: application to abdominal oxygen mapping.

A novel imaging modality is introduced which uses radiofrequency longitudinally detected electron spin resonance (RF-LODESR). It is capable of providing qualitative and semiquantitative information on a variety of parameters reflecting physiological function, the most significant being tissue oxygenation. Effective spin-lattice (T*1e) and spin-spin (T*2e) electronic relaxation time maps of the abdomen of living 200-g rats were generated after intravenous administration of a triarylmethyl free radical (TAM). These maps were used to evaluate oxygen distribution. Differences between the liver, kidneys, and bladder were noted. Conclusions were made regarding the distribution, perfusion, and excretion rate of the contrast medium. Ligature-induced anoxia in the kidney was also visualized. LODESR involves transverse ESR irradiation with a modulated excitation, and observing oscillations in the spin magnetization parallel to the main magnetic field. The T*1e and T*2e maps were calculated from a set of LODESR signal phase images collected at different detection frequencies. Each phase image also provides qualitative information on tissue oxygen levels without any further processing. This method presents an alternative to the conventional transverse ESR linewidth-based oximetry methods, particularly for animal whole-body imaging applications.     

适用于微型CT系统活体成像的新型门控技术

目的微型CT(Micro-CT)具有解剖成像及无损伤的特点,但对软组织分辨率较低,且在活体成像中,动物呼吸和心脏运动会引入伪影。为提高图像质量,本文提出一种新型门控技术。方法门控技术通过检测动物的心肺运动,在其某个生理时相产生门控信号,并触发X射线球管曝光,最终将获得的投影图像用于三维重建。心肺门控系统主要由监控软件,数字信号处理器(DSP),心电(ECG)检测电路及热电偶电路组成。结果在小鼠活体实验中,获取了6组采用不同门控信号得到的CT投影数据。结论实验结果表明,心肺门控技术的使用,极大地提高了小动物在自由呼吸状态下,使用Micro-CT系统进行活体成像的图像分辨率。     

Comparison of methods for reduction of lipid contamination for in vivo proton MR spectroscopic imaging of the brain.

In vivo proton MR spectroscopic imaging (MRSI) of human brain is complicated by the presence of a strong signal from subcutaneous lipids, which may result in signal contamination in metabolite images obtained following Fourier-transform reconstruction. In this study, two approaches for reduction of lipid contamination--using postprocessing and additional data acquisition--are compared. The first uses extrapolation of k-space information for subcutaneous lipid, which has been applied to data obtained using conventional fully phase-encoded MRSI with circularly sampled k-space or echo-planar spectroscopic imaging (EPSI). The second uses a dual EPSI technique that combines multiple-averaged central k-space data with a single EPSI acquisition of additional information that is used for improved lipid reconstruction. Comparisons are carried out with data obtained from human brain in vivo at 1.5 T with short and medium TEs. Results demonstrate that the performance of both methods for reducing the effects of lipid contamination is similar, and that both are limited by the effects of instrumental instabilities and subject motion, which also depend on the acquisition method used.     

Spatially resolved time-course studies of free radical reactions with an EPRI/MRI fusion technique.

Electron paramagnetic resonance imaging (EPRI) using nitroxyl radicals is a useful technique for visualizing reactive oxygen species (ROS) and the pharmacokinetics of probes. To unambiguously identify anatomical locations, coregistration of EPRI with images obtained by MRI is necessary. In this study the feasibility of performing reliable EPRI/MRI fusion imaging using nitroxyl radical fiducial markers was tested. The pharmacokinetics of the nitroxyl radicals were observed after oral or intravenous administration in C57BL6 mice. To fuse both images, the nitroxyl radical was used as fiducial markers. The EPR and MR images corresponded well and clearly illustrated minimal changes in pharmacokinetics between carbamoyl-PROXYL and carboxy-PROXYL. These results demonstrate that the EPRI/MRI fused imaging technique is useful for investigating in vivo pharmacokinetics and provides unambiguous anatomic details.