Multisite EPR oximetry from multiple quadrature harmonics

Multisite continuous wave (CW) electron paramagnetic resonance (EPR) oximetry using multiple quadrature field modulation harmonics is presented. First, a recently developed digital receiver is used to extract multiple harmonics of field modulated projection data. Second, a forward model is presented that relates the projection data to unknown parameters, including linewidth at each site. Third, a maximum likelihood estimator of unknown parameters is reported using an iterative algorithm capable of jointly processing multiple quadrature harmonics. The data modeling and processing are applicable for parametric lineshapes under nonsaturating conditions. Joint processing of multiple harmonics leads to 2-3-fold acceleration of EPR data acquisition. For demonstration in two spatial dimensions, both simulations and phantom studies on an L-band system are reported.     

Nano-emulsions of fluorinated trityl radicals as sensors for EPR oximetry

This article reports the development and evaluation of two nano-emulsions (F45T-03/HFB and F15T-03/PFOB) containing fluorinated trityl radicals dissolved in perfluorocarbons. Preparation with a high-pressure homogenizer conferred sub-micronic size to both nano-emulsions. In vitro and in vivo EPR spectroscopy showed that the nano-emulsions had much greater oxygen sensitivity than the hydrophilic trityl, CT-03. In vivo experiments in rodents confirmed the ability of the nano-emulsions to follow the changes in oxygen concentration after induced ischemia. Histological evaluation of the tissue injected with the nano-emulsions revealed some acute toxicity for the F45T-03/HFB nano-emulsion but none for the F15T-03/PFOB nano-emulsion. These new formulations should be considered for further EPR oximetry experiments in pathophysiological situations where subtle changes in tissue oxygenation are expected.     

Development of isoindoline nitroxides for EPR oximetry in viable systems

Nitroxides are widely used as biophysical probes to study molecular motion, intracellular oxygen, pH, transmembrane potential, and cellular redox metabolism, etc. They may be rapidly metabolized to hydroxylamines by cells, which limits their use in viable systems. In this study, we have characterized relevant properties in cells of several isoindoline nitroxides that have been prepared to have different physicochemical properties: 1,1,3,3-tetramethylisoindolin-2-yloxyl (TMIO) and its analogs 5-carboxy-1,1,3,3-tetramethylisoindolin-2-yloxyl (CTMIO), 5-(N,N,N-trimethylammonio)-1,1,3,3-tetramethyl isoindolin-2-yloxyl iodide (QATMIO) and 2-hydroxy-1,1,3,3-tetramethylisoindoline hydrochloride (TMIOH.HCI). The oxygen sensitivity and metabolic kinetics of these were compared in CHO cells under different oxygen tensions with 1-oxyl-2,2,6,6-tetramethyl-4-piperidione (Tempone) and 3-carboxyl-2,2,5,5-tetramethyl-pyrrolidine-1-oxyl (PCA). Cytotoxicity was evaluated by the measurement of oxygen consumption rates, trypan blue exclusion, and clone formation. TMIO and its analogues have a higher relative oxygen sensitivity than Tempone and PCA with the oxygen sensitivity in electron paramagnetic resonance (EPR) spectrometry in the order of: TMIO=TMIOH=CTMIO>QATMIO=Tempone<PCA. The rates of metabolism of these nitroxides are moderate and depend on oxygen concentration, ring type, ring substituent, and membrane permeation. These nitroxides have low cytotoxicity. The results indicate that TMIO and its analogues are potentially useful for EPR studies of viable systems, especially for oximetry.     

Optimization of magnetic field sweep and field modulation amplitude for continuous-wave EPR oximetry

For continuous-wave electron paramagnetic resonance spectroscopy, what settings of magnetic field sweep width and field modulation amplitude yield the best accuracy in estimated linewidth? Statistical bounds on estimation error presented in this work provide practical guidance: set the sweep width and modulation amplitude to 8 and 4 times the half-width half-maximum linewidth, Γ, respectively. For unknown linewidths in the range [Γ(min),Γ(max)] the worst-case estimation error is minimized by using settings designed for Γ(max). The analysis assumes a Lorentzian lineshape and a constant modulation amplitude across the extent of the irradiated paramagnetic probe. The analytical guidelines are validated using L-band spectroscopy with a particulate LiNc-BuO probe.     

Concentration by centrifugation for gas exchange EPR oximetry measurements with loop-gap resonators

Measurement of the bimolecular collision rate between a spin label and oxygen is conveniently carried out using a gas permeable plastic sample tube of small diameter that fits a loop-gap resonator. It is often desirable to concentrate the sample by centrifugation in order to improve the signal-to-noise ratio (SNR), but the deformable nature of small plastic sample tubes presents technical problems. Solutions to these problems are described. Two geometries were considered: (i) a methylpentene polymer, TPX, from Mitsui Chemicals, at X-band and (ii) Teflon tubing with 0.075 mm wall thickness at Q-band. Sample holders were fabricated from Delrin that fit the Eppendorf microcentrifuge tubes and support the sample capillaries. For TPX, pressure of the sealant at the end of the sample tube against the Delrin sample holder provided an adequate seal. For Teflon, the holder permitted introduction of water around the tube in order to equalize pressures across the sealant during centrifugation. Typically, the SNR was improved by a factor of five to eight. Oxygen accessibility applications in site-directed spin labeling studies are discussed.     

Evaluation of oxygen-response times of phthalocyanine-based crystalline paramagnetic spin probes for EPR oximetry

The goal of the present study was to evaluate the temporal response of particulate-based EPR oximetry probes to changes in partial pressure of oxygen (pO(2)). In order to accurately evaluate the oxygen-response time, we developed a method for rapid modulation of pO(2) in a chamber containing the probe using an oscillator-driven speaker-diaphragm setup. The apparatus was capable of producing sinusoidal changes in pO(2) at frequencies up to 300 Hz or more. The pressure-modulation setup was used to evaluate the temporal response of some of the most commonly used phthalocyanine-based particulate probes. For validation, the time-response of the probes was compared to that of a high sensitivity pressure sensor. The results revealed that some particulate probes could respond to changes in pO(2) with a temporal response of 3.3 ms (300 Hz). The observations were interpreted in the light of their crystalline packing in favor of oxygen diffusion. The results of the present study should enable the selection of probes for oximetry applications requiring high temporal resolution.     

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.     

The evaluation of new and isotopically labeled isoindoline nitroxides and an azaphenalene nitroxide for EPR oximetry

Isoindoline nitroxides are potentially useful probes for viable biological systems, exhibiting low cytotoxicity, moderate rates of biological reduction and favorable Electron Paramagnetic Resonance (EPR) characteristics. We have evaluated the anionic (5-carboxy-1,1,3,3-tetramethylisoindolin-2-yloxyl; CTMIO), cationic (5-(N,N,N-trimethylammonio)-1,1,3,3-tetramethylisoindolin-2-yloxyl iodide, QATMIO) and neutral (1,1,3,3-tetramethylisoindolin-2-yloxyl; TMIO) nitroxides and their isotopically labeled analogs ((2)H(12)- and/or (2)H(12)-(15)N-labeled) as potential EPR oximetry probes. An active ester analogue of CTMIO, designed to localize intracellularly, and the azaphenalene nitroxide 1,1,3,3-tetramethyl-2,3-dihydro-2-azaphenalen-2-yloxyl (TMAO) were also studied. While the EPR spectra of the unlabeled nitroxides exhibit high sensitivity to O(2) concentration, deuteration resulted in a loss of superhyperfine features and a subsequent reduction in O(2) sensitivity. Labeling the nitroxides with (15)N increased the signal intensity and this may be useful in decreasing the detection limits for in vivo measurements. The active ester nitroxide showed approximately 6% intracellular localization and low cytotoxicity. The EPR spectra of TMAO nitroxide indicated an increased rigidity in the nitroxide ring, due to dibenzo-annulation.     

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.     

Theoretical study of spin-temperature phenomena in EPR for the low-temperature approximation and steady-state conditions

Using the concept of spin temperature in EPR in the low-temperature approximation and under steady-state conditions, we examine the dependence of the reciprocal spin temperature of the Zeeman subsystem and the dipole-dipole reservoir on the reciprocal lattice temperature in the presence of a saturating field. We show that more significant cooling of the dipole-dipole reservoir and heating of the Zeeman subsystem can be achieved than in the case of the high-temperature approximation. In the presence of a probe field, we study the conditions for the absorption to go to zero at the frequency of this field, as a function of the intensity of the saturating field and the frequency difference between the two fields. We show that the intensity of the saturating field increases as the frequencies of the fields come closer together, and goes to infinity when they coincide. This qualitatively corresponds to what we have in the case of the high-temperature approximation. The intensity of the saturating field has a minimum in the dependence on detuning of the frequency of the saturating field when the frequency of the probe field is fixed, as in the case of the high-temperature approximation. We can use this dependence to estimate the relaxation parameters. __________ Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 73, No. 1, pp. 82–85, January–February, 2006.     

Practical limitations to accuracy in a nulling automatic wavelength-scanning ellipsometer

The limits of accuracy in an automatic wavelength-scanning ellipsometer are defined on the basis of measured mechanical properties of the instrument and the geometrical and optical defects of the optical components. A computational procedure is described for obtaining accurate Δ and ψ values by using a matrix model of the instrument, and the areás in which the greatest improvements in accuracy can be achieved are identified.     

Single-molecule force spectroscopy: Practical limitations beyond Bell’s model

Single-molecule force spectroscopy experiments, and a number of other physical systems, are governed by thermally activated transitions out of a metastable state under the action of a steadily increasing external force. The main observable in such experiments is the distribution of the forces, at which the escape events occur. The challenge in interpreting the experimental data is to relate them to the microscopic system properties. We work out a maximum likelihood approach and show that it is the optimal method to tackle this problem. When fitting actual experimental data it is unavoidable to assume some functional form for the force-dependent escape rate. Focusing on escape processes over a single activation barrier, we consider a quite general and common such functional form and demonstrate by means of data from a realistic computer experiment that the maximum number of fit parameters that can be determined reliably is three. They are related to the force-free escape rate and the position and height of the activation barrier. Furthermore, the results for the first two of these fit parameters show little dependence on the assumption about the manner in which the barrier decreases with the applied force, while the last one, the barrier height in the absence of force, depends strongly on this assumption.     

X射线高空间分辨多色显微成像系统研制及应用

在激光间接驱动惯性约束聚变(ICF)领域中,获得具有极高空间分辨率(优于5μm)的X射线辐射图像,是研究烧蚀不稳定性、内爆流线等关键物理过程的数据基础。基于掠入射反射式成像原理的Kirkpatrick-Baez(KB)显微成像系统作为一种具有高空间分辨率和集光效率的X射线显微诊断设备,目前已成为国际ICF装置的X射线关键诊断设备。在神光Ⅱ和神光Ⅲ原型装置条件上开展了KB诊断技术及设备的研究,在KB系统的光学设计、光学元件和物镜与系统装调技术等方面取得了许多重要进展,研制了大视场KB、多色KB等高分辨率X射线显微成像系统。这些系统已应用于我国的ICF内爆芯部发光和流线测量、流体不稳定增长测量等实验中,为关键物理量的测量提供了高空间分辨率的清晰图像。     

X射线高空间分辨多色显微成像系统研制及应用

在激光间接驱动惯性约束聚变（ICF）领域中,获得具有极高空间分辨率（优于5 m）的X射线辐射图像,是研究烧蚀不稳定性、内爆流线等关键物理过程的数据基础。基于掠入射反射式成像原理的Kirkpatrick-Baez(KB)显微成像系统作为一种具有高空间分辨率和集光效率的X射线显微诊断设备,目前已成为国际ICF装置的X射线关键诊断设备。在神光Ⅱ和神光Ⅲ原型装置条件上开展了KB诊断技术及设备的研究,在KB系统的光学设计、光学元件和物镜与系统装调技术等方面取得了许多重要进展,研制了大视场KB、多色KB等高分辨率X射线显微成像系统。这些系统已应用于我国的ICF内爆芯部发光和流线测量、流体不稳定增长测量等实验中,为关键物理量的测量提供了高空间分辨率的清晰图像。     

Vectorized multisite coding for hydrodynamic cellular automata

Simulating eight lattices for Pomeau's cellular automata simultaneously through bit-per-bit operations, a vectorized Fortran program reached 30 million updates per second and per Cray YMP processor. We give the full innermost loops.     

The noise immunity of gradiometer coils for14N NQR land mine detection: Practical limitations

Magnetic resonance measurements in the field, such as for land mine detection using¹⁴N nuclear quadrupole resonance (NQR), must deal with large levels of environmental radio-frequency interference. One approach to minimize the level of interference which enters the NQR receiver is the use of a coil with no magnetic dipole moment. Such a coil is, ideally, sensitive only to spatial gradients of the magnetic fields and is referred to as a gradiometer. It is straightforward to estimate the amount of reduction one can expect for an ideal gradiometer. Here it is shown that for¹⁴N NQR land mine detection in practice, the ultimate level of interference received can be expected to be significantly greater than what one would expect solely due to these spatial gradients. This is due to the fact that it is quite difficult to construct an ideal gradiometer.     

The aqueous reference for ESR oximetry

The interaction of molecular oxygen with derivatives of nitroxide EPR spin labels has been investigated using nuclear spin-relaxation spectroscopy in aqueous and nonaqueous solvents. The proton spin-lattice relaxation rate induced by oxygen provides a measure of the local concentration of oxygen, which we find is dependent on solvent. In water, the hydrophobic effect increases the local concentration of oxygen in the nonpolar portions of solute molecules. For nitroxides reduced to the hydroxylamine in aqueous solutions, we find that the local concentration of oxygen is approximately twice that associated with a free diffusion hard sphere limit, while in octane, this effect is absent. These results show that nitroxide based ESR oximetry may suffer a reference concentration shift of order a factor of two if the aqueous nitroxide spectrum or relaxation is used as the reference.     

Quantitative EPR — Sensitivity to experimental conditions and optimal setting of recording parameters

This is the proportionality of the area under undistorted absorption curve to the spin number contributing to the resonance phenomena which makes the electron paramagnetic resonance spectrometry a method of quantitative analysis. However, quantitative EPR (Q-EPR) is not so simple as the relation is. Direct quantitative measurements are practically excluded, the comparison of both standard and unknown sample EPR signals (indirect determination) is rather used, as a rule. The accuracy of spin number determination is an essential problem of quantitative EPR. The experimental conditions and instrumental variables influencing the signal shape and intensity may lower the accuracy of quantitative determinations. There is different sensitivity of various line parameters to the instrumental distortions and experimental conditions. Thus, the optimal setting of instrumental variables controlling the distortions depends on the procedure being used for quantitative determinations. The aspects of sensitivity of different Q-EPR procedures to instrumental distortions, as well as the rules of optimal setting of these parameters are the subject of this review.     

Planar microresonators for EPR experiments

EPR resonators on the basis of standing-wave cavities are optimised for large samples. For small samples it is possible to design different resonators that have much better power handling properties and higher sensitivity. Other parameters being equal, the sensitivity of the resonator can be increased by minimising its size and thus increasing the filling factor. Like in NMR, it is possible to use lumped elements; coils can confine the microwave field to volumes that are much smaller than the wavelength. We discuss the design and evaluation of EPR resonators on the basis of planar microcoils. Our test resonators, which operate at a frequency of 14 GHz, have excellent microwave efficiency factors, achieving 24 ns pi/2 EPR pulses with an input power of 17 mW. The sensitivity tests with DPPH samples resulted in the sensitivity value 2.3 x 10(9) spins.G(-1) Hz(-1/2) at 300 K.