Variable radio frequency proton-electron double-resonance imaging: application to pH mapping of aqueous samples

Proton-electron double-resonance imaging (PEDRI) offers rapid image data collection and high resolution for spatial distribution of paramagnetic probes. Recently we developed the concept of variable field (VF) PEDRI which enables extracting a functional map from a limited number of images acquired at pre-selected EPR excitation fields using specific paramagnetic probes (Khramtsov et al., J. Magn. Reson. 202 (2010) 267-273). In this work, we propose and evaluate a new modality of PEDRI-based functional imaging with enhanced temporal resolution which we term variable radio frequency (VRF) PEDRI. The approach allows for functional mapping (e.g., pH mapping) using specifically designed paramagnetic probes with high quality spatial resolution and short acquisition times. This approach uses a stationary magnetic field but different EPR RFs. The ratio of Overhauser enhancements measured at each pixel at two different excitation frequencies corresponding to the resonances of protonated and deprotonated forms of a pH-sensitive nitroxide is converted to a pH map using a corresponding calibration curve. Elimination of field cycling decreased the acquisition time by exclusion periods of ramping and stabilization of the magnetic field. Improved magnetic field homogeneity and stability allowed for the fast MRI acquisition modalities such as fast spin echo. In total, about 30-fold decrease in EPR irradiation time was achieved for VRF PEDRI (2.4s) compared with VF PEDRI (70s). This is particularly important for in vivo applications enabling one to overcome the limiting stability of paramagnetic probes and sample overheating by reducing RF power deposition.     

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.     

Low-Field, Time-Resolved Dynamic Nuclear Polarization with Field Cycling and High-Resolution NMR Detection

Dynamic nuclear polarization (DNP) effects in aqueous solutions of stable 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) radicals were studied in a pulsed mode of pumping the electron paramagnetic resonance (EPR) transitions. Our fast field cycling setup allowed us to perform the EPR pumping at low magnetic fields and to detect the enhanced nuclear magnetic resonance signals at 7 T with high spectral resolution. Pumping was performed at two different frequencies, 300 MHz and 1.4 GHz, corresponding to magnetic fields around 10 and 48.6 mT, respectively. For both frequencies, the DNP enhancements were close to the limiting theoretical values of ?110 (¹⁴N TEMPOL) and ?165 (¹⁵N TEMPOL). Our pulsed experiments exploit coherent motion of the electronic spins in the radio-frequency field as seen by an oscillatory component in the dependence of the DNP effect on the radio-frequency pulse duration. The DNP enhancement was studied in detail as a function of the pulse length, duty cycle, delay between the pulses, and applied power. The analysis of the results shows that pulsed DNP experiments provide an opportunity to achieve enhancements of about ?110 with relatively low applied power as compared to the standard continuous-wave DNP experiments. An adequate theoretical approach to the problem under study is suggested.     

Radiation Dosimetry Using Alanine and Electron Paramagnetic Resonance (EPR) Spectroscopy: A New Look at an Old Topic

The detection and quantification by electron paramagnetic resonance (EPR) spectroscopy of stable radicals formed in alanine by exposure to γ-radiation is used as a secondary standard for radiation dosimetry measurements, even though the EPR signal is actually derived from >1 radical with different spectral properties. For high radiation doses, microwave power saturation and spectral linewidths are both dependent on the received dose, and result in non-linear calibration curves. Furthermore, using a high-sensitivity microwave cavity, the power at which EPR signal saturation commences is ~0.3–0.4 mW for samples with irradiation doses ≤10 kGy; these values are an order of magnitude lower than those normally used in alanine dosimetry. In addition, the central peak of the first derivative spectrum, the height of which is commonly used in dosimetry measurements, is the most susceptible to microwave power saturation. Therefore, for high-level dosimetry we now recommend that analyses be performed under non-saturating conditions, and that the spectral acquisition parameters should be determined with a standard irradiated to ≤10 kGy to eliminate any intensity problems associated with variable saturation characteristics. At low radiation doses, variations in spectral saturation characteristics are negligible, and partially saturating conditions along with modulation amplitudes much higher than those normally used can reliably produce improved signal-to-noise ratios and allow extension of the methodology to practical working limits of ~0.1–0.2 Gy.     

Study of Stable L-Alanine Radicals by W-Band EPR Spectroscopy

Stable L-alanine radicals, SAR1 and SAR2, induced by γ-irradiation of the L-alanine crystal have been investigated by electron paramagnetic resonance (EPR) technique at W-band (94 GHz) frequency. The study provides assignment of radical centers detected by continuous-wave EPR, saturation transfer mode and echo-detected field-swept EPR at W-band frequencies. The phase memory time, T _m, which was measured simultaneously at X-band (9.5 GHz) and W-band frequencies for different spectral components has been employed to estimate rotation correlation times of CH₃ protons and an effective correlation time related to the local dynamics of the entire SAR1 center at room temperature.     

Object dependent sweep width reduction with spectral-spatial EPR imaging

For spectral-spatial EPR imaging, prior knowledge about the spatial support of an imaged object can be exploited in two ways. We can shrink the spatial field of view (FOV) to closely wrap the object in a sphere or reduce the sweep width in a projection dependent fashion. Use of a smaller spatial FOV with the same number of samples enhances spatial resolution by reducing voxel volume at the expense of signal-to-noise and a consequent degraded line-width resolution. We have developed another approach to define sweep width that prunes away the portions of the projection sweep with no signal. This reduces data acquisition time for the continuous wave (CW) EPR image proportional to the sweep width reduction. This method also avoids voxel volume reduction. Using the reduced-sweep method, we decreased the data acquisition time by 20% maintaining spatial and linewidth resolution.     

Feasibility of Q-Band EPR Dosimetry in Biopsy Samples of Dental Enamel, Dentine and Bone

Electron paramagnetic resonance (EPR) dosimetry of tooth enamel in X-band has been established as a suitable method for individual reconstruction of doses 0.1 Gy and higher. The objective was to demonstrate the feasibility of using Q-band EPR in small biopsy tooth enamel samples to provide accurate measurements of radiation doses. Q-band spectra of small (<10 mg) irradiated samples of dentine and bone were studied to investigate the possibility of using Q-band EPR for dose measurements in those materials if there are limited amounts of enamel available, and there is no time for the chemical sample preparation required for accurate X-band measurements in dental enamel. Our results have shown that Q-band provides accurate measurements of radiation doses higher than 0.5 Gy in tooth enamel biopsy samples as small as 2 mg. Q-band EPR spectra in powdered dentine and bone demonstrated significantly higher resolution and sensitivity than in conventional X-band measurements.     

Biochemical EPR imaging of skin

EPR Imaging (EPRI) of spin labels is a powerful method for investigating skin and can give information about biochemical processes which are involved in numerous skin diseases. Furthermore it enables the non invasive investigation of the liberation, penetration and distribution of spin labelled drugs. The basis of these measurements is spectral spatial EPR imaging employing modulated field gradients and simultaneous field scans (MOSS). A skin region (?=6 mm) was treated with a 10 μl spin label solution (1 mM). EPR spectra of 128 points were recorded in 128 spatial planes resulting in a 128×128 image matrix. A spatial resolution of better than 10 μm can be obtained for a spectral line width of 0.1 mT and a gradient of 4 Tm^?1.In vivo imaging on mammalian skin can be performed by employing surface coils at S-band frequencies, 3 GHz.     

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.     

Parallel-plate RF resonator imaging of chemical shift resolved capillary flow

Magnetic resonance imaging has been introduced to study flow in microchannels using pure phase spatial encoding with a microfabricated parallel-plate nuclear magnetic resonance (NMR) probe. The NMR probe and pure phase spatial encoding enhance the sensitivity and resolution of the measurement. In this paper, ¹H NMR spectra and images were acquired at 100 MHz. The B₁ magnetic field is homogeneous and the signal-to-noise ratio of 30 μl doped water for a single scan is 8×10⁴. The high sensitivity of the probe enables velocity mapping of the fluids in the micro-channel with a spatial resolution of 13×13 μm. The parallel-plate probe with pure phase encoding permits the acquisition of NMR spectra; therefore, chemical shift resolved velocity mapping was also undertaken. Results are presented which show separate velocity maps for water and methanol flowing through a straight circular micro-channel. Finally, future performance of these techniques for the study of microfluidics is extrapolated and discussed.     

Rapid acquisition strategy for functional T1ρ mapping of the brain

Functional T1ρ mapping has been proposed as a method to assess pH and metabolism dynamics in the brain with high spatial and temporal resolution. The purpose of this work is to describe and evaluate a variant of the spin-locked echo-planar imaging sequence for functional T1ρ mapping at 3 T. The proposed sequence rapidly acquires a time series of T1ρ maps with 4.0 second temporal resolution and 10 slices of volumetric coverage. Simulation, phantom, and in vivo experiments are used to evaluate many aspects of the sequence and its implementation including fidelity of measured T1ρ dynamics, potential confounds to the T1ρ response, imaging parameter tradeoffs, time series analysis approaches, and differences compared to blood oxygen level dependent functional magnetic resonance imaging. It is shown that the high temporal resolution and volumetric coverage of the sequence are obtained with some expense including underestimation of the T1ρ response, sensitivity to T1 dynamics, and reduced signal-to-noise ratio. In vivo studies using a flashing checkerboard functional magnetic resonance imaging paradigm suggest differences between T1ρ and blood oxygen level dependent activation patterns. Possible sources of the functional T1ρ response and potential sequence improvements are discussed. The capability of T1ρ to map whole-brain pH and metabolism dynamics with high temporal and spatial resolution is potentially unique and warrants further investigation and development.     

SSFP fMRI at 3 tesla: Efficiency of polar acquisition-reconstruction technique

SSFP-based fMRI techniques, known for their high specificity and low geometrical distortion, look promising for high-resolution brain mapping. Nevertheless, they suffer from lack of speed and sensitivity, leading them to be exploited mostly in high-field scanners. Radial acquisition can help with these inefficiencies through better tSNR and more effective coverage of the spatial frequencies. Here, we present a SSFP-fMRI approach and experimentally investigate it at 3 T scanners using radial readout for acquisition. In particular, the visual activity is mapped through three bSSFP techniques: 1- Cartesian, 2- Radial with re-gridding reconstruction, 3- Radial with Polar Fourier Transform (PFT) reconstruction. In the PFT technique streaking artifacts, generated at high acceleration rates by re-gridding reconstruction, are avoided and pixel size in the final framework is retrospectively selectable. General agreement, but better tSNR of Radial reading, was first confirmed for these techniques in detection of neural activities at 2 × 2 mm² in-plane resolution for all 28 subjects,. Next the outcome of the PFT algorithm with 1 × 1 mm² pixel size was compared to images reconstructed by re-gridding (from the same raw data) with the identical pixel size through interpolation. The localization of the activity showed improvement in PFT over interpolation both qualitatively (i.e., well-fitting in gray-matter) and quantitatively (i.e., higher z-scores and tSNR). The proposed technique can therefore be considered as a remedy for lack of speed and sensitivity in SSFP-based fMRI, in conventional field strengths. The proposed approach is particularly useful in task-based studies when we concentrate on a ROI considerably smaller than FOV, without sacrificing spatial resolution.     

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.     

Advances in constant-velocity Mössbauer instrumentation

A prototype of a programmable constant-velocity scaler is presented. This instrument allows the acquisition of partial Mössbauer spectra in selected energy regions using standard drivers and transducers. It can be fully operated by a remote application, thus data acquisition can be automated. The instrument consists of a programmable counter and a constant-velocity reference. The reference waveform generator is amplitude modulated with 13-bit resolution, and is programmable in a wide range of frequencies and waveforms in order to optimize the performance of the transducer. The counter is compatible with most standard SCA, and is configured as a rate-meter that provides counts per selectable time slice at the programmed velocity. As a demonstration of the instrument applications, a partial Mössbauer spectrum of a natural iron foil was taken. Only positive energies were studied in 512 channels, accumulating 20 s per channel. A line width of 0.20 mm/s was achieved, performing with an efficiency of 80%.     

Spin-label W-band EPR with Seven-Loop–Six-Gap Resonator: Application to Lens Membranes Derived from Eyes of a Single Donor

Spin-label W-band (94 GHz) electron paramagnetic resonance (EPR) with a five-loop–four-gap resonator (LGR) was successfully applied to study membrane properties (Mainali et al. J Magn Reson 226:35–44, 2013). In that study, samples were equilibrated with the selected gas mixture outside the resonator in a sample volume ~100 times larger than the sensitive volume of the LGR and transferred to the resonator in a quartz capillary. A seven-loop–six-gap W-band resonator has been developed. This resonator permits measurements on aqueous samples of 150 nL volume positioned in a polytetrafluoroethylene (PTFE) gas permeable sample tube. Samples can be promptly deoxygenated or equilibrated with an air/nitrogen mixture inside the resonator, which is significant in saturation-recovery measurements and in spin-label oximetry. This approach was tested for lens lipid membranes derived from lipids extracted from two porcine lenses (single donor). Profiles of membrane fluidity and the oxygen transport parameter were obtained from saturation-recovery EPR using phospholipid analog spin-labels. Cholesterol analog spin-labels allowed discrimination of the cholesterol bilayer domain and acquisition of oxygen transport parameter profiles across this domain. Results were compared with those obtained previously for membranes derived from a pool of 100 lenses. Results demonstrate that EPR at W-band can be successfully used to study aqueous biological samples of small volume under controlled oxygen concentration.     

Multi-Frequency EPR and DC Conductivity of Itinerant Spins in Single-Wall Carbon Nanotubes

Sealed, deoxygenated single-wall carbon nanotubes show two characteristic electron paramagnetic resonance (EPR) signals at g = 2.07 and g = 2.00 in the temperature range from 300 to 50 K. Reversible interconversion between both components was observed. The large g-shift and the temperature dependence of the EPR susceptibility of the g = 2.07 signal indicate that this signal can be attributed to itinerant spins. At low temperatures only the g = 2.00 signal remained, which could be further characterized using microwave frequencies up to 320 GHz. The direct current conductivity of a partially aligned sample was also measured. The room temperature value was estimated as 0.7 (Ωcm)^?1. The observed temperature dependence can be described by assuming temperature-activated hopping in a small-gap semiconductor with an activation energy of 3.5 meV, similar to the characteristics of the previously measured 9.4 GHz microwave conductivity.     

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.     

High spatial resolution BOLD fMRI using simultaneous multislice excitation with echo-shifting gradient echo at 7 Tesla

We introduce an accelerated gradient echo (GRE) sequence combining simultaneous multislice excitation (SMS) with echo-shifting technique for high spatial resolution blood oxygen level dependent (BOLD) functional MRI (fMRI). The simulation was conducted to optimize scan parameters. To validate the feasibility of the proposed technique, the visual and motor task experiments were performed at 7.0 Tesla (T). The single-shot EPI sequence was also applied in comparison with the proposed technique. The simulation results showed that an optimized flip angle of 9° provided maximal BOLD contrast for our scanning scheme, allowing low power deposition and SMS acceleration factor of 5. Additionally, parallel acquisition imaging with acceleration factor of 2 was utilized, which allowed a total acceleration factor of 10 in volunteer study. The experiment results showed that geometric distortion-free BOLD images with voxel size of 1.0 × 1.0 × 2.5 mm³ were obtained. Significant brain activation was identified in both visual and motor task experiments, which were in accordance with previous investigations. The proposed technique has potential for high spatial resolution fMRI at ultra-high field because of its sufficient BOLD sensitivity as well as improved acquisition speed over conventional GRE-based techniques.     

Progressive EPR imaging with adaptive projection acquisition

Continuous wave electron paramagnetic resonance imaging (EPRI) of living biological systems requires rapid acquisition and visualization of free radical images. In the commonly used multiple-stage back-projection image reconstruction algorithm, the EPR image cannot be reconstructed until a complete set of projections is collected. If the data acquisition is incomplete, the previously acquired incomplete data set is no longer useful. In this work, a 3-dimensional progressive EPRI technique was implemented based on inverse Radon transform in which a 3-dimensional EPR image is acquired and reconstructed gradually from low resolution to high resolution. An adaptive data acquisition strategy is proposed to determine the significance of projections and acquire them in an order from the most significant to the least significant. The image acquisition can be terminated at any time if further collection of projections does not improve the image resolution distinctly, providing flexibility to trade image quality with imaging time. The progressive imaging technique was validated using computer simulations as well as imaging experiments. The adaptive acquisition uses 50-70% less projections as compared to the regular acquisition. In conclusion, adaptive data acquisition with progressive image reconstruction should be very useful for the accelerated acquisition and visualization of free radical distribution.     

Intracellular pH mapping with SNARF-1 and confocal microscopy. I: A quantitative technique for living tissue and isolated cells

A fluorescent pH indicator in conjunction with confocal microscopy, was used to map intracellular pH in a variety of cells and tissues with high spatial resolution. The new pH-sensitive fluorescent probe SNARF-1 was excited with the 488 nm band of the argon ion laser of a Bio-Rad MRC-500 confocal microscope. Ratio images were created with pixel-by-pixel division, with the intensity of these images representing a function of pH, that is independent of dye concentration, photobleaching or path length. Cell cultures of rat aortic smooth muscle were loaded with 20 μм SNARF-1/AM for 20 min at 37°C. Intracellular pH levels were calibrated in situ by treatment of each cell with nigericin (20 μм) in solutions of known pH. The cytosolic pH of the majority of cells was uniform, however, pH gradients were evident between the cytosol and nuclear regions, indicating the ability of this technique to map intracellular and intraorganelle pH. Rat C6 glioblastoma spheroids were cultured then loaded with SNARF-1/AM at 10°C for 90 min. The pH values were calibrated in vitro, using SNARF-1 acid in buffered solutions of known pH. Ratio images of the bisected spheroids showed a marked gradient in pH from the outer cells compared with central necrotic cells. The degree of involvement of acidification in muscle fatigue was investigated by simultaneously determining force generation and intracellular pH in individual fibres of an intact rat muscle. The investigation was performed during a stimulation protocol which induced significant fatigue in the force response of the muscle. The fatigue protocol induced little change in cytosolic pH in the fibres. We show that the use of SNARF-1, in conjunction with confocal microscopy is a powerful technique for accurately mapping pH within single cells, multicellular tissues and intact organs, as well as for accurately recording dynamic changes in pH.