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

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 1.25 mm. 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.     

Proton-Electron Double-Resonance Imaging of pH Using Phosphonated Trityl Probe

Variable radio frequency proton-electron double-resonance imaging (VRF PEDRI) enables extracting a functional map from a limited number of images acquired at pre-selected EPR frequencies using specifically designed paramagnetic probes with high-quality spatial resolution and short acquisition times. In this work we explored the potential of VRF PEDRI for pH mapping of aqueous samples using recently synthesized pH-sensitive phosphonated trityl radical, pTR. The ratio of Overhauser enhancements measured at each pixel at two different excitation frequencies corresponding to the resonances of protonated and deprotonated forms of pTR probe allows for a pH map extraction. Long relaxation times of pTR allow for pH mapping at EPR irradiation power as low as 1.25 W during 130 s acquisition time with spatial resolution of about 1 mm. This is particularly important for in vivo applications enabling one to avoid sample overheating by reducing RF power deposition.     

Fast 3D spatial EPR imaging using spiral magnetic field gradient

Electron paramagnetic resonance imaging (EPRI) provides direct detection and mapping of free radicals. The continuous wave (CW) EPRI technique, in particular, has been widely used in a variety of applications in the fields of biology and medicine due to its high sensitivity and applicability to a wide range of free radicals and paramagnetic species. However, the technique requires long image acquisition periods, and this limits its use for many in vivo applications where relatively rapid changes occur in the magnitude and distribution of spins. Therefore, there has been a great need to develop fast EPRI techniques. We report the development of a fast 3D CW EPRI technique using spiral magnetic field gradient. By spiraling the magnetic field gradient and stepping the main magnetic field, this approach acquires a 3D image in one sweep of the main magnetic field, enabling significant reduction of the imaging time. A direct one-stage 3D image reconstruction algorithm, modified for reconstruction of the EPR images from the projections acquired with the spiral magnetic field gradient, was used. We demonstrated using a home-built L-band EPR system that the spiral magnetic field gradient technique enabled a 4-7-fold accelerated acquisition of projections. This technique has great potential for in vivo studies of free radicals and their metabolism.     

Standard-based method for proton-electron double resonance imaging of oxygen

Proton-electron double resonance imaging (PEDRI) has been utilized for indirect determination of oxygen concentrations in aqueous samples and living systems. Due to the complexity of the problem, there are seven oxygen related parameters that need to be measured to determine the distribution of oxygen. We present an improved approach in which image intensities from only two PEDRI acquisitions with different EPR irradiation powers are required to determine the distribution of a paramagnetic probe and oxygen in an analyzed sample. This is achieved using three reference samples with known concentrations of a paramagnetic probe and oxygen placed inside the resonator together with the measurement sample. An EPR-off image, which has low signal intensity at low magnetic field (0.02 T) is not required for the calculations, significantly reducing the total time of the experiments and the noise while enhancing the accuracy of these oxygen measurements. The Finland trityl radical was used as the paramagnetic probe and oxygen concentrations could be accurately measured and imaged over the physiological range from 0 to 240 μM.     

Reporting of quantitative oxygen mapping in EPR imaging

Oxygen maps derived from electron paramagnetic resonance spectral-spatial imaging (EPRI) are based upon the relaxivity of molecular oxygen with paramagnetic spin probes. This technique can be combined with MRI to facilitate mapping of pO(2) values in specific anatomic locations with high precision. The co-registration procedure, which matches the physical and digital dimensions of EPR and MR images, may present the pO(2) map at the higher MRI resolution, exaggerating the spatial resolution of oxygen, making it difficult to precisely distinguish hypoxic regions from normoxic regions. The latter distinction is critical in monitoring the treatment of cancer by radiation and chemotherapy, since it is well-established that hypoxic regions are three or four times more resistant to treatment compared to normoxic regions. The aim of this article is to describe pO(2) maps based on the intrinsic resolution of EPRI. A spectral parameter that affects the intrinsic spatial resolution of EPRI is the full width at half maximum (FWHM) height of the gradient-free EPR absorption line in frequency-encoded imaging. In single point imaging too, the transverse relaxation times (T(2)(?)) limit the resolution since the signal decays by exp(-t(p)/T(2)(?)) where the delay time after excitation pulse, t(p), is related to the resolution. Although the spin densities of two point objects may be resolved at this separation, it is inadequate to evaluate quantitative changes of pO(2) levels since the linewidths are proportionately affected by pO(2). A spatial separation of at least twice this resolution is necessary to correctly identify a change in pO(2) level. In addition, the pO(2) values are blurred by uncertainties arising from spectral dimensions. Blurring due to noise and low resolution modulates the pO(2) levels at the boundaries of hypoxic and normoxic regions resulting in higher apparent pO(2) levels in hypoxic regions. Therefore, specification of intrinsic resolution and pO(2) uncertainties are necessary to interpret digitally processed pO(2) illustrations.     

Development and testing of a CW-EPR apparatus for imaging of short-lifetime nitroxyl radicals in mouse head

This article describes a method for reducing the acquisition time in three-dimensional (3D) continuous-wave electron paramagnetic resonance (CW-EPR) imaging. To visualize nitroxyl spin probes, which have a short lifetime in living organisms, the acquisition time for a data set of spectral projections should be shorter than the lifetime of the spin probes. To decrease the total time required for data acquisition, the duration of magnetic field scanning was reduced to 0.5 s. Moreover, the number of projections was decreased by using the concept of a uniform distribution. To demonstrate this faster data acquisition, two kinds of nitroxyl radicals with different decay rates were measured in mice. 3D EPR imaging of 4-hydroxy-2,2,6,6-tetramethylpiperidine-d₁₇-1-¹⁵N-1-oxyl in mouse head was successfully carried out. 3D EPR imaging of nitroxyl spin probes with a half-life of a few minutes was achieved for the first time in live animals.     

Magic-angle sample spinning electron paramagnetic resonance--instrumentation, performance, and limitations

An electron paramagnetic resonance (EPR) setup for line narrowing experiments with fast sample spinning at variable angles between the rotation axis and the static magnetic field is described and applied in the magic-angle sample spinning (MAS) EPR experiment at X-band frequencies (9.5 GHz). Sample spinning speeds up to 17 kHz at temperatures down to 200 K can be achieved with rotors of 4-mm outer and 2.5-mm inner diameter without severe losses in microwave amplitude compared to standard pulse EPR probeheads. A phase cycle is introduced that provides pure absorption MAS EPR spectra and allows one to distinguish between positive and negative frequency offsets (pseudo-quadrature detection). Possible broadening mechanisms in MAS EPR spectra are discussed. It is demonstrated both by theory and by experiment that the MAS EPR experiment requires excitation bandwidths that are comparable to the total spectral width, since otherwise destructive interference between contributions of spins with similar resonance offsets suppresses the signal. Experimental observations on the E(1) center in gamma-irradiated silica glass and on the SO(-)(3) radical in gamma-irradiated sulfamic acid are reported.     

Electron paramagnetic resonance imaging using magnetic-field-gradient spinning

A novel X-band CW EPR imaging has been developed using magnetic-field-gradient (MFG) spinning to obtain spatial distributions of electron paramagnetic species. Spinning MFG EPR imaging for 65 projection spectra required just 55 s while conventional imaging took 11 min 40 s, that is, the acquisition time for the new system is one order of magnitude shorter than that for conventional EPR imaging. Spinning MFG EPR imaging allows one to measure reconstructed images in an interactive manner where resolution and condition can be changed quickly.     

Estimation of the in vivo decay rate of EPR signals for a nitroxide radical in rat brains by a region-selected intensity determination method

The region-selected intensity determination (RSID) method was proposed to obtain the temporal changes in electron paramagnetic resonance (EPR) signal intensity from a selected region by a stationary magnetic field gradient. To select the region, the subtraction field that was derived from the distance between the center and the projection of the selected region to the direction of the field gradient was applied to the main field. The directions of the stationary magnetic field gradient at a constant strength were systematically changed in a three-dimensional space after each acquisition of the spectrum. All spectra under the field gradient were accumulated and the resultant spectrum was deconvoluted by a spectrum without the field gradient. The center height of the deconvoluted spectrum indicates the signal intensity of the selected region. To verify this method, a phantom or in vivo study was conducted on a 700 MHz radio-frequency EPR spectrometer equipped with a bridged loop-gap resonator. In the temporal EPR measurements of phantoms including a nitroxide radical aqueous solution with and without ascorbic acid, the selected regions were alternatively changed at the position of the two phantoms. The signal intensity derived from the one phantom showed an exponential decay, and for the other phantom, no temporal changes. The spatial resolution of this method was estimated to be 2.7 mm by using a pinpoint phantom that included diphenylpicrylhydrazyl powder. In the in vivo temporal EPR measurements, the selected regions were alternatively changed at the cerebral cortex and the striatum of rats that had received a blood-brain barrier-permeative nitroxide radical. The decay rate of the signal intensity at each region obtained by this method was consistent with those previously reported.     

Simultaneous acquisition of pulse EPR orientation selective spectra

High resolution pulse EPR methods are usually applied to resolve weak magnetic electron-nuclear or electron-electron interactions that are otherwise unresolved in the EPR spectrum. Complete information regarding different magnetic interactions, namely, principal components and orientation of principal axis system with respect to the molecular frame, can be derived from orientation selective pulsed EPR measurements that are performed at different magnetic field positions within the inhomogeneously broadened EPR spectrum. These experiments are usually carried out consecutively, namely a particular field position is chosen, data are accumulated until the signal to noise ratio is satisfactory, and then the next field position is chosen and data are accumulated. Here we present a new approach for data acquisition of pulsed EPR experiments referred to as parallel acquisition. It is applicable when the spectral width is much broader than the excitation bandwidth of the applied pulse sequence and it is particularly useful for orientation selective pulse EPR experiments. In this approach several pulse EPR measurements are performed within the waiting (repetition) time between consecutive pulse sequences during which spin lattice relaxation takes place. This is achieved by rapidly changing the main magnetic field, B(0), to different values within the EPR spectrum, performing the same experiment on the otherwise idle spins. This scheme represents an efficient utilization of the spectrometer and provides the same spectral information in a shorter time. This approach is demonstrated on W-band orientation selective electron-nuclear double resonance (ENDOR), electron spin echo envelope modulation (ESEEM), electron-electron double resonance (ELDOR)--detected NMR and double electron-electron resonance (DEER) measurements on frozen solutions of nitroxides. We show that a factors of 3-6 reduction in total acquisition time can be obtained, depending on the experiment applied.     

High spatial resolution multi-site EPR oximetry. The use of convolution-based fitting method

We describe a new method to enhance the spatial resolution of multi-site electron paramagnetic resonance (EPR) oximetry. The method is suitable for any shape (density distribution function) of a solid paramagnetic material implanted in tissue. It corrects distortions of lineshapes caused by the gradient and thus overcomes limitations of previous multi-site EPR oximetry methods that restricted the ratio of the particle size to the distance between sites. The new method is based on consecutive applications of magnetic field gradients with the same direction but with a different magnitude and uses a convolution-based fitting algorithm to derive Lorentzian EPR linewidths of each individual peak of the EPR spectrum. The method is applicable for any particulate EPR oxygen sensitive materials whose EPR spectra can be approximated by a Lorentzian function or a superposition of Lorentzian functions. By incorporating this model of the lineshape in the data processing, we are able to decrease significantly the number of parameters needed for the calculations and to recover the oxygen concentration, even from quite noisy spectra. We (i) describe our method and the data-processing algorithm, (ii) demonstrate our approach in model and in vivo experiments, and (iii) discuss the limitations.     

A new strategy for fast radiofrequency CW EPR imaging: direct detection with rapid scan and rotating gradients

Rapid field scan on the order of T/s using high frequency sinusoidal or triangular sweep fields superimposed on the main Zeeman field, was used for direct detection of signals without low-frequency field modulation. Simultaneous application of space-encoding rotating field gradients have been employed to perform fast CW EPR imaging using direct detection that could, in principle, approach the speed of pulsed FT EPR imaging. The method takes advantage of the well-known rapid-scan strategy in CW NMR and EPR that allows arbitrarily fast field sweep and the simultaneous application of spinning gradients that allows fast spatial encoding. This leads to fast functional EPR imaging and, depending on the spin concentration, spectrometer sensitivity and detection band width, can provide improved temporal resolution that is important to interrogate dynamics of spin perfusion, pharmacokinetics, spectral spatial imaging, dynamic oxymetry, etc.     

EPR oximetry in three spatial dimensions using sparse spin distribution

A method is presented to use continuous wave electron paramagnetic resonance imaging for rapid measurement of oxygen partial pressure in three spatial dimensions. A particulate paramagnetic probe is employed to create a sparse distribution of spins in a volume of interest. Information encoding location and spectral linewidth is collected by varying the spatial orientation and strength of an applied magnetic gradient field. Data processing exploits the spatial sparseness of spins to detect voxels with nonzero spin and to estimate the spectral linewidth for those voxels. The parsimonious representation of spin locations and linewidths permits an order of magnitude reduction in data acquisition time, compared to four-dimensional tomographic reconstruction using traditional spectral-spatial imaging. The proposed oximetry method is experimentally demonstrated for a lithium octa-n-butoxy naphthalocyanine (LiNc–BuO) probe using an L-band EPR spectrometer.     

Real-time monitoring of drug-induced changes in the stomach acidity of living rats using improved pH-sensitive nitroxides and low-field EPR techniques

New improved pH-sensitive nitroxides were applied for in vivo studies. An increased stability of the probes towards reduction was achieved by the introduction of the bulky ethyl groups in the vicinity of the paramagnetic NO fragment. In addition, the range of pH sensitivity of the approach was extended by the synthesis of probes with two ionizable groups, and, therefore, with two pKa values. Stability towards reduction and spectral characteristics of the three new probes were determined in vitro using 290 MHz radiofrequency (RF)- and X-band electron paramagnetic resonance (EPR), longitudinally detected EPR (LODEPR), and field-cycled dynamic nuclear polarization (FC-DNP) techniques. The newly synthesized probe, 4-[bis(2-hydroxyethyl)amino]-2-pyridine-4-yl-2,5,5-triethyl-2,5-dihydro-1H-imidazol-oxyl, was found to be the most appropriate for the application in the stomach due to both higher stability and convenient pH sensitivity range from pH 1.8 to 6. LODEPR, FC-DNP and proton-electron double resonance imaging (PEDRI) techniques were used to detect the nitroxide localization and acidity in the rat stomach. Improved probe characteristics allowed us to follow in vivo the drug-induced perturbation in the stomach acidity and its normalization afterwards during 1 h or longer period of time. The results show the applicability of the techniques for monitoring drug pharmacology and disease in the living animals.     

High Spatial Resolution Multi-Site EPR Oximetry: The Use of a Convolution-Based Fitting Method

We describe a new method to enhance the spatial resolution of multi-site electron paramagnetic resonance (EPR) oximetry. The method is suitable for any shape (density distribution function) of a solid paramagnetic material implanted in tissue. It corrects distortions of lineshapes caused by the gradient and thus overcomes limitations of previous multi-site EPR oximetry methods that restricted the ratio of the particle size to the distance between sites. The new method is based on consecutive applications of magnetic field gradients with the same direction but with a different magnitude and uses a convolution-based fitting algorithm to derive Lorentzian EPR linewidths of each individual peak of the EPR spectrum. The method is applicable for any particulate EPR oxygen sensitive materials whose EPR spectra can be approximated by a Lorentzian function or a superposition of Lorentzian functions. By incorporating this model of the lineshape in the data processing, we are able to decrease significantly the number of parameters needed for the calculations and to recover the oxygen concentration, even from quite noisy spectra. We (i) describe our method and the data-processing algorithm, (ii) demonstrate our approach in model and in vivo experiments, and (iii) discuss the limitations.     

Recent developments in high frequency/high magnetic field CW EPR. Applications in chemistry and biology

A recent review of developments in high magnetic field/high frequency continuous wave electron paramagnetic resonance (EPR) spectroscopy is presented. The main motivations to develop instrumentation beyond “conventional” spectrometers are the desire to increase the spectral resolution and the need for high frequencies in the case of large zero field splitting systems. The potential of high frequency EPR is illustrated with the study of a Mn based spin cluster, which also confirms the need for multifrequency measurements. Examples of high field/high frequency EPR applications to chemistry and biology demonstrate that additional structural information can be obtained with the resolution achieved at 245 GHz.     

Enhanced resolution for EPR imaging by two-step deblurring

The broad spectrum of spin probes used for electron paramagnetic resonance imaging (EPRI) result in poor spatial resolution of the reconstructed images. Conventional deconvolution procedures can enhance the resolution to some extent but obtaining high resolution EPR images is still a challenge. In this work, we have implemented and analyzed the performance of a postacquisition deblurring technique to enhance the spatial resolution of the EPR images. The technique consists of two steps; noniterative deconvolution followed by iterative deconvolution of the acquired projections which are then projected back using filtered backprojection (FBP) to reconstruct a high resolution image. Further, we have proposed an analogous technique for iterative reconstruction algorithms such as multiplicative simultaneous iterative reconstruction technique (MSIRT) which can be a method of choice for many applications. The performance of the suggested deblurring approach is evaluated using computer simulations and EPRI experiments. Results suggest that the proposed procedure is superior to the standard FBP and standard iterative reconstruction algorithms in terms of mean-square-error (MSE), spatial resolution, and visual judgment. Although the procedure is described for 2D imaging, it can be readily extended to 3D imaging.     

A high spatial resolution 1H magnetic resonance spectroscopic imaging technique for breast cancer with a short echo time

The high sensitivity but low specificity of breast MRI has prompted exploration of breast (1)H MRS for breast cancer detection. However, several obstacles still prevent the routine application of in vivo breast (1)H MRS, including poor spatial resolution, long acquisition time associated with conventional multi-voxel MRS imaging (MRSI) techniques, and the difficulty of "extra" lipid suppression in a magnetic field with relatively poor achievable homogeneity compared to the brain. Using a combination of a recently developed echo-filter (EF) suppression technique and an elliptical sampling scheme, we demonstrate the feasibility of overcoming these difficulties. It is robust (the suppression technique is insensitive to magnetic field inhomogeneity), fast (acquisition time of about 12 min) and offers high spatial resolution (up to 0.6 cm(3) per voxel at 1.5 T with a TE of only 60 ms). This approach should be even better at 3 T with higher resolution and/or shorter TE.