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.     

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.     

Imaging spin probe distribution in the tumor of a living mouse with 250 MHz EPR: correlation with BOLD MRI.

Electron paramagnetic resonance imaging (EPRI) promises to provide new insights into the physiology of tissues in health and disease. Understanding the in vivo imaging capability of this new modality requires comparison with other physiologically responsive techniques. Here, an initial comparison between 2D EPR spatial imaging of a narrow single line injectable paramagnetic trityl spin probe and 2D slice-selected carbogen subtraction BOLD MRI is presented. The images were obtained from the same FSa fibrosarcoma grown in the leg of a C3H mouse. This tumor was unusual in comparison with others imaged with subtraction BOLD MRI because of its peripheral distribution of intensity. The spatial distribution of the EPR spin probe showed the same peripheral distribution. The pixel resolutions of these images are comparable. These images provide an early in vivo comparison of EPRI with a well-established imaging modality. The comparison validates the in vivo distribution of spin probe as imaged with EPRI, and provides a proof of principle for the comparison of BOLD and 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.     

Evaluation of the dose distribution gradient in the close vicinity of brachytherapy seeds using electron paramagnetic resonance imaging

Electron paramagnetic resonance (EPR) spectroscopy has been successfully employed to determine radiation dose using alanine. The EPR signal intensity reflects the number of stable free radicals produced, and provides a quantitative measurement of the absorbed dose. The aim of the present study was to explore whether this principle can be extended to provide information on spatial dose distribution using EPR imaging (EPRI). Lithium formate was selected because irradiation induces a single EPR line, a characteristic that is particularly convenient for imaging purposes. ¹²⁵I‐brachytherapy seeds were inserted in tablets made of lithium formate. Images were acquired at 1.1 GHz. Monte Carlo (MC) calculations were used for comparison. The dose gradient can be determined using two‐dimensional (2D) EPR images. Quantitative data correlated with the dose estimated by the MC simulations, although differences were observed. This study provides a first proof‐of‐concept that EPRI can be used to estimate the gradient dose distribution in phantoms after irradiation. Magn Reson Med, 2009. © 2009 Wiley‐Liss, Inc.     

Development of a hybrid EPR/NMR coimaging system.

Electron paramagnetic resonance imaging (EPRI) is a powerful technique that enables spatial mapping of free radicals or other paramagnetic compounds; however, it does not in itself provide anatomic visualization of the body. Proton magnetic resonance imaging (MRI) is well suited to provide anatomical visualization. A hybrid EPR/NMR coimaging instrument was constructed that utilizes the complementary capabilities of both techniques, superimposing EPR and proton-MR images to provide the distribution of paramagnetic species in the body. A common magnet and field gradient system is utilized along with a dual EPR and proton-NMR resonator assembly, enabling coimaging without the need to move the sample. EPRI is performed at approximately 1.2 GHz/ approximately 40 mT and proton MRI is performed at 16.18 MHz/ approximately 380 mT; hence the method is suitable for whole-body coimaging of living mice. The gradient system used is calibrated and controlled in such a manner that the spatial geometry of the two acquired images is matched, enabling their superposition without additional postprocessing or marker registration. The performance of the system was tested in a series of phantoms and in vivo applications by mapping the location of a paramagnetic probe in the gastrointestinal (GI) tract of mice. This hybrid EPR/NMR coimaging instrument enables imaging of paramagnetic molecules along with their anatomic localization in the body.     

Feasibility and assessment of non-invasive in vivo redox status using electron paramagnetic resonance imaging

Purpose:
To test the feasibility of electron paramagnetic resonance imaging (EPRI) to provide non-invasive images of tissue redox status using redox-sensitive paramagnetic contrast agents. Material and Methods:
Nitroxide free radicals were used as paramagnetic agents and a custom-built 300 MHz EPR spectrometer/imager was used for all studies. A phantom was constructed consisting of four tubes containing equal concentrations of a nitroxide. Varying concentrations of hypoxanthine/xanthine oxidase were added to each tube and reduction of the nitroxide was monitored by EPR as a function of time. Tumor-bearing mice were intravenously infused with a nitroxide and the corresponding reduction rate was monitored on a pixel-by-pixel basis using 2D EPR of the tumor-bearing leg and normal leg serving as control. For animal studies, nitroxides were injected intravenously (1.25 mmol/kg) and EPR projections were collected every 3 min after injection using a magnetic field gradient of 2.5 G/cm. The reduction rates of signal intensity on a pixel-by-pixel basis were calculated and plotted as a redox map. Redox maps were also collected from the mice treated with diethylmaleate (DEM), which depletes tissue thiols and alters the global redox status. Results:
Redox maps obtained from the phantoms were in agreement with the intensity change in each of the tubes where the signals were decreasing as a function of the enzymatic activity, validating the ability of EPRI to accurately access changes in nitroxide reduction. Redox imaging capability of EPR was next evaluated in vivo. EPR images of the nitroxide distribution and reduction rates in tumor-bearing leg of mice exhibited more heterogeneity than in the normal tissue. Reduction rates were found to be significantly decreased in tumors of mice treated with DEM, consistent with the depletion of thiols and the consequent alteration of the redox status. Conclusion:
Using redox-sensitive paramagnetic contrast agents, EPRI can non-invasively discriminate redox status differences between normal tissue and tumors.     

Advantageous application of a surface coil to EPR irradiation in overhauser‐enhanced MRI

The present study describes the advantageous application of a surface coil to electron paramagnetic resonance (EPR) irradiation in Overhauser‐enhanced MRI (OMRI). OMRI is a double‐resonance method for imaging free radicals based on the Overhauser effect. Proton NMR images are recorded without and with EPR irradiation of the free radical resonance, which results in a difference proton image that shows signal enhancement in spatial regions that contain the free radical. To obtain good signal enhancement in OMRI, very high RF power and a long EPR irradiation time are required. To improve sensitivity and shorten the image acquisition time, especially for localized (and topical) applications, we developed and tested a surface‐coil‐type EPR irradiation coil. Theoretical calculations and experimental data showed that EPR irradiation through the surface coil could ameliorate the localized Overhauser enhancement, which was related to the ratio of B₁ surface coil/B₁ volume coil in the region of interest (ROI), as expected. The increased sensitivity could also be converted into a shortened EPR irradiation time, resulting in fast data acquisition. For biomedical applications, the use of a surface coil (as opposed to a conventional volume coil) could decrease the total RF power deposition in the sample required to obtain the same Overhauser enhancement in the ROI. Magn Reson Med 57:806–811, 2007. © 2007 Wiley‐Liss, Inc.     

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).     

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.     

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.     

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.     

In vivo proton electron double resonance imaging of mice with fast spin echo pulse sequence

Purpose:

To develop and evaluate a two‐dimensional (2D) fast spin echo (FSE) pulse sequence for enhancing temporal resolution and reducing tissue heating for in vivo proton electron double resonance imaging (PEDRI) of mice.

Materials and Methods:

A four‐compartment phantom containing 2 mM TEMPONE was imaged at 20.1 mT using 2D FSE‐PEDRI and regular gradient echo (GRE)‐PEDRI pulse sequences. Control mice were infused with TEMPONE over ～1 min followed by time‐course imaging using the 2D FSE‐PEDRI sequence at intervals of 10–30 s between image acquisitions. The average signal intensity from the time‐course images was analyzed using a first‐order kinetics model.

Results:

Phantom experiments demonstrated that EPR power deposition can be greatly reduced using the FSE‐PEDRI pulse sequence compared with the conventional gradient echo pulse sequence. High temporal resolution was achieved at ～4 s per image acquisition using the FSE‐PEDRI sequence with a good image SNR in the range of 233–266 in the phantom study. The TEMPONE half‐life measured in vivo was ～72 s.

Conclusion:

Thus, the FSE‐PEDRI pulse sequence enables fast in vivo functional imaging of free radical probes in small animals greatly reducing EPR irradiation time with decreased power deposition and provides increased temporal resolution. J. Magn. Reson. Imaging 2012;471‐475. © 2011 Wiley Periodicals, Inc.     

Automatic slice positioning (ASP) for passive real‐time tracking of interventional devices using projection‐reconstruction imaging with echo‐dephasing (PRIDE)

A novel and fast approach for passive real‐time tracking of interventional devices using paramagnetic markers, termed “projection‐reconstruction imaging with echo‐dephasing” (PRIDE) is presented. PRIDE is based on the acquisition of echo‐dephased projections along all three physical axes. Dephasing is preferably set to 4π within each projection ensuring that background tissues do not contribute to signal formation and thus appear heavily suppressed. However, within the close vicinity of the paramagnetic marker, local gradient fields compensate for the intrinsic dephasing to form an echo. Successful localization of the paramagnetic marker with PRIDE is demonstrated in vitro and in vivo in the presence of different types of off‐resonance (air/tissue interfaces, main magnetic field inhomogeneities, etc). In order to utilize the PRIDE sequence for vascular interventional applications, it was interleaved with balanced steady‐state free precession (bSSFP) to provide positional updates to the imaged slice using a dedicated real‐time feedback link. Active slice positioning (ASP) with PRIDE is demonstrated in vitro, requiring approximately 20 ms for the positional update to the imaging sequence, comparable to existing active tracking methods. Magn Reson Med, 2009. © 2009 Wiley‐Liss, Inc.     

In vivo imaging of changes in tumor oxygenation during growth and after treatment.

A novel procedure for in vivo imaging of the oxygen partial pressure (pO2) in implanted tumors is reported. The procedure uses electron paramagnetic resonance imaging (EPRI) of oxygen-sensing nanoprobes embedded in the tumor cells. Unlike existing methods of pO2 quantification, wherein the probes are physically inserted at the time of measurement, the new approach uses cells that are preinternalized (labeled) with the oxygen-sensing probes, which become permanently embedded in the developed tumor. Radiation-induced fibrosarcoma (RIF-1) cells, internalized with nanoprobes of lithium octa-n-butoxy-naphthalocyanine (LiNc-BuO), were allowed to grow as a solid tumor. In vivo imaging of the growing tumor showed a heterogeneous distribution of the spin probe, as well as oxygenation in the tumor volume. The pO2 images obtained after the tumors were exposed to a single dose of 30-Gy X-radiation showed marked redistribution as well as an overall increase in tissue oxygenation, with a maximum increase 6 hr after irradiation. However, larger tumors with a poorly perfused core showed no significant changes in oxygenation. In summary, the use of in vivo EPR technology with embedded oxygen-sensitive nanoprobes enabled noninvasive visualization of dynamic changes in the intracellular pO2 of growing and irradiated tumors.     

Fast three dimensional sodium imaging

An efficient scheme for fast three dimensional acquisition of sodium MR images is described. This scheme relies on the use of three dimensional k-space trajectories with constant sample density to achieve significant (60–70%) reductions in total data acquisition time over conventional projection imaging schemes. The performance of this data acquisition scheme is demonstrated with acquisition of sodium data sets on phantoms and normal human volunteers at 1.5 and 3.0 Tesla. The experimental results demonstrate that high quality three dimensional sodium images (0.2 cc voxel size, 10:1 signal-to-noise ratio) can be acquired at clinical field strengths (1.5 Tesla) in under 10 min.     

Three-dimensional spatial EPR imaging of the rat heart

While instrumentation capable of performing three-dimensional EPR imaging of free radicals in whole tissues and isolated organs has been developed at L-band, important questions remain regarding the resolution and image quality that can be obtained in practice using the presently available free radical labels. Therefore, studies were performed applying three-dimensional spatial EPR imaging at L-band to image the distribution of free radical labels in the isolated heart and in phantoms of similar size. With nitroxide labels the obtainable resolution is limited by the presence of hyperfine structure in the EPR absorption function that in turn limits the maximum applicable gradient. The authors observed that with the nitroxide labels, resolutions in the range of 1–2 mm are possible, while with a single line glucose char label, resolutions of 0.2 mm are obtained. With the nitroxides, images were of sufficient resolution to resolve the overall global shape of the heart and the location of the left and right ventricular cavities; however, finer structures could not be resolved. With the glucose char much finer resolution could be obtained enabling visualization of the ventricles, aortic root, and proximal coronary arteries.     

Split‐acquisition real‐time CINE phase‐contrast MR flow measurements

The temporal and spatial resolution of real‐time phase‐contrast magnetic resonance (PCMR) is restricted by the need to acquire two interleaved phase images. In this article, we propose a split‐acquisition real‐time CINE PCMR technique, where the acquisition of flow‐encoded and flow‐compensated data is divided into separate blocks. By comparing magnitude images, automatic matching of data in cardio‐respiratory space allows subtraction of background phase offsets. Thus, the data is acquired in real‐time but with phase correction originating from a different heart beat. This effectively doubles the frame rate, allowing either higher temporal or spatial resolution. Two split‐acquisition sequences were tested: one with high‐temporal resolution and one with high‐spatial resolution. Both sequences showed excellent agreement in stroke volumes in 20 adults when validated against cardiac‐gated PCMR and interleaved real‐time PCMR (cardiac gated: 95.2 ± 20.0 mL, interleaved real‐time: 96.2 ± 20.7 mL, high‐temporal resolution: 95.6 ± 20.1 mL, high‐spatial resolution: 95.5 ± 20.4 mL). In six children, the high‐spatial resolution sequence provided more accurate flow measurements than interleaved real‐time PCMR, when compared with cardiac‐gated PCMR (cardiac gated: 20.6 ± 7.6 mL, interleaved real‐time: 24.3 ± 9.2 mL, high‐spatial resolution: 20.8 ± 7.8 mL), due to the increased spatial resolution. The matching technique is shown to be accurate (truth: 94.6 ± 21.8, split‐acquisition: 95.0 ± 21.9 mL) and quantitative image quality (signal‐to‐noise ratio, velocity‐to‐noise ratio and edge sharpness) is acceptable. Magn Reson Med, 2010. © 2010 Wiley‐Liss, Inc.     

Fast 3D 1H spectroscopic imaging at 3 Tesla using spectroscopic missing-pulse SSFP with 3D spatial preselection.

Three-dimensional (3D) (1)H MR spectroscopic imaging (SI) allows metabolic changes in human tissue to be identified. In clinical practice, fast acquisition techniques are required to achieve an adequate spatial resolution within acceptable total measurement times. In this study a novel fast pulse sequence for 3D (1)H SI based on the condition of steady-state free precession (SSFP), termed "spectroscopic missing-pulse SSFP" (spMP-SSFP), is proposed. It combines 3D spatial preselection with the acquisition of full spin echoes (SEs), and thus makes subsequent phase correction of spectra redundant. The sequence was applied to a phantom and healthy human brains in vivo at 3 Tesla. Metabolic images are acquired with a spatial resolution of 1.8 cm(3) within a total measurement time of about 6 min. With a lower signal-to-noise ratio (SNR) per unit measurement time compared to previous spectroscopic SSFP implementations, 3D spatial preselection can now be realized with spMP-SSFP. Since the method does not require separate techniques for water and lipid suppression, and employs a simple data-processing approach, spMP-SSFP is a robust, fast SI method that requires only minimal user interaction.     

In vivo EPR spectroscopic imaging for a liposomal drug delivery system.

We used the membrane-impermeable nitroxyl radical 4-trimethylammonium-2,2,6,6-tetramethylpiperidine-1-oxyliodide (CAT-1) as a model drug encapsulated in liposomes in order to separately map the 2D distribution of both liposomal-encapsulated CAT-1 and free CAT-1. Phantoms were prepared with a CAT-1 solution and a liposomal CAT-1 suspension. Spectral-spatial images were obtained along several polar-arranged spatial axes through the phantom. The 1D spatial distributions (projections) of each signal component, reflecting the concentration of CAT-1, were then extracted from the spectral-spatial images. 2D EPR images of liposomal-encapsulated CAT-1 and free CAT-1 were separately reconstructed from the resulting projection data sets. 2D mapping of each component exhibited good agreement with respect to the phantom. Separate maps were generated from separate injections of free CAT-1 and liposomal CAT-1 injected into the femoral muscle of a living mouse. The EPR signal of the free CAT-1 gradually decreased during data acquisition. Because of this decay, we calibrated the image intensity by extrapolating the signal intensity to that detected at the beginning of data sampling. Both the position and size of the individual images were in very good agreement with those of the mouse thigh obtained by MRI.