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In JoVE (1)
Other Publications (15)
- Spectrochimica Acta. Part A, Molecular and Biomolecular Spectroscopy
- Journal of Magnetic Resonance (San Diego, Calif. : 1997)
- Journal of the American College of Cardiology
- Magnetic Resonance in Medicine : Official Journal of the Society of Magnetic Resonance in Medicine / Society of Magnetic Resonance in Medicine
- Journal of Controlled Release : Official Journal of the Controlled Release Society
- Physical Chemistry Chemical Physics : PCCP
- Magnetic Resonance in Medicine : Official Journal of the Society of Magnetic Resonance in Medicine / Society of Magnetic Resonance in Medicine
- Journal of Magnetic Resonance (San Diego, Calif. : 1997)
- Biosensors & Bioelectronics
- Physical Chemistry Chemical Physics : PCCP
- Biophysical Journal
- The Review of Scientific Instruments
- The Review of Scientific Instruments
- The Review of Scientific Instruments
- Biophysical Journal
Articles by Aharon Blank in JoVE
JoVE General
Electron Spin Resonance Micro-imaging of Live Species for Oxygen Mapping
Schulich Faculty of Chemistry, The Technion, Israel Institute of Technology
This protocol describes a method for micron-scale three-dimensional imaging of oxygen concentration in the immediate environment of live cells by electron spin resonance microscopy.
Other articles by Aharon Blank on PubMed
Filling Factor in a Pulsed Electron Paramagnetic Resonance Experiment
A definition and mathematical treatment to calculate the filling factor in a pulsed electron paramagnetic resonance (EPR) experiment are presented. The differences between filling factors in traditional, continuous wave (CW)-EPR experiments (eta), and in pulsed-EPR experiments (eta(p)), are discussed. We present some examples to demonstrate how eta(p) depends upon the particular pulse sequence and sample characteristics.
High Resolution Electron Spin Resonance Microscopy
NMR microscopy is routinely employed in fields of science such as biology, botany, and materials science to observe magnetic parameters and transport phenomena in small scale structures. Despite extensive efforts, the resolution of this method is limited (>10 microm for short acquisition times), and thus cannot answer many key questions in these fields. We show, through theoretical prediction and initial experiments, that ESR microscopy, although much less developed, can improve upon the resolution limits of NMR, and successfully undertake the 1 mum resolution challenge. Our theoretical predictions demonstrate that existing ESR technology, along with advanced imaging probe design (resonator and gradient coils), using solutions of narrow linewidth radicals (the trityl family), should yield 64 x 64 pixels 2D images (with z slice selection) with a resolution of 1 x 1 x 10 microm at approximately 60 GHz in less than 1h of acquisition. Our initial imaging results, conducted by CW ESR at X-band, support these theoretical predictions and already improve upon the previously reported state-of-the-art for 2D ESR image resolution achieving approximately 10 x 10 mum, in just several minutes of acquisition time. We analyze how future progress, which includes improved resonators, increased frequency of measurement, and advanced pulsed techniques, should achieve the goal of micron resolution.
Diagnosis of Thin-cap Fibroatheromas by a Self-contained Intravascular Magnetic Resonance Imaging Probe in Ex Vivo Human Aortas and in Situ Coronary Arteries
We sought to correlate findings obtained from a self-contained magnetic resonance imaging (MRI) probe with plaque morphology of ex vivo human aortas and coronary arteries.
Miniature Self-contained Intravascular Magnetic Resonance (IVMI) Probe for Clinical Applications
A miniature (1.73 mm in diameter) NMR probe, which contains a magnet and a radiofrequency (RF) coil, is presented. This probe is integrated at the tip of a standard catheter and can be inserted into the human coronary arteries, creating local magnetic fields needed to obtain the NMR signal from the blood vessel walls, without the need for external magnet or RF coils. The basic theory governing the probe performance in terms of signal-to-noise-ratio and contrast parameters is presented, along with measured results from test samples. The NMR signal can be analyzed to obtain tissue contrast parameters such as T1, T2 and the diffusion coefficient, which may be used to detect lipid-rich vulnerable plaques in the coronary arteries.
Electron Spin Resonance Microscopy Applied to the Study of Controlled Drug Release
We describe our recent developments towards 3D micron-scale imaging capability, based on electron spin resonance (ESR), and its application to the study of controlled release. The method, termed ESR microscopy (ESRM), is an extension of the conventional "millimeter-scale" ESR imaging technique. It employs paramagnetic molecules (such as stable radicals or spin-labeled drugs) and may enable one to obtain accurate 3D spatially resolved information about the drug concentration, its self-diffusion tensor, rotational correlation time and the pH in the release matrix. Theoretical calculations, along with initial experimental results, suggest that a 3D resolution of approximately 1 microm is feasible with this method. Here we were able to image successfully a high spin concentration sample with a resolution of approximately 3 x 3 x 8 microm and subsequently study a single approximately 120 microm biodegradable microsphere, internalized with a dilute solution of trityl radical, with a resolution of approximately 12.7 x 13.2 x 26 microm. Analysis of the microsphere ESR imaging data revealed a likely increase in the viscosity inside the sphere and/or binding of the radical molecule to the sphere matrix. Future directions for progress are also discussed.
ESR Imaging in Solid Phase Down to Sub-micron Resolution: Methodology and Applications
Electron spin resonance microcopy (ESRM) is an imaging method aimed at the observation of paramagnetic species in small samples with micron-scale spatial resolution. At present, this technique is pursued mainly for biological applications at room temperature and in relatively low static magnetic fields. This work is focused on the use of ESRM for the measurement of solid samples. First, a brief comparison of various electron spin resonance (ESR) detection techniques is provided, with an emphasis on conventional "induction detection". Following that, some methodological details are provided along with experimental examples carried out at room temperature and in a static field of approximately 0.5 T. These examples show for the first time the imaging of solid samples measured by "induction detection" ESR with a resolution better than 1 mum. Based on these experimental examples and capabilities, an outlook for the future prospects of this methodology in terms of spin sensitivity and resolution is provided. It is estimated that single-spin sensitivity could be achieved for some samples at liquid-helium temperatures and static fields of approximately 2 T. Furthermore, under these conditions, spatial resolution could reach the nanometer scale. Finally, a description of possible applications of this new methodology is provided.
Ex Situ Endorectal MRI Probe for Prostate Imaging
A unique ex situ MRI probe, which examines samples external to its geometry, is presented. The probe is intended to be used for imaging the prostate gland via an endorectal pathway. It has a semicylindrical shape with a length of 6 cm and typical diameter of approximately 3 cm. The probe's imaging field of view spans almost along its entire length and up to a distance of 2 cm away from its surface, with an angular sector of approximately 90 degrees . The detailed design of the probe is presented, followed by a set of representative results obtained by the current bench prototype of this system.
ESR Micro-imaging of LiNc-BuO Crystals in PDMS: Spatial and Spectral Grain Distribution
Microcrystals of lithium octa-n-butoxynaphthalocyanine (LiNc-BuO) in a bio-compatible and oxygen-permeable polymer matrix of poly-dimethyl-siloxane (PDMS) can be used for repetitive non-invasive imaging of oxygen in live specimens by means of mm-scale electron spin resonance (ESR) imaging. This probe denoted as "oxychip" was characterized by high-resolution mum-scale ESR microcopy to reveal the fine details of its spatial and spectral properties. The ESR micro-images of a typical oxychip device showed that while the spatial distribution of the microcrystals in the polymer is fairly homogenous (as revealed by optical microscopy), the ESR signal originates only from a very few dominant crystals. Furthermore, spectral-spatial analysis in a microcrystal and a sub-microcrystal spatial resolution reveals that each crystal has a slightly different g-factor and also exhibits variations in linewidth, possibly due to the slightly different individual crystallization process.
A Molecular Paramagnetic Spin-doped Biopolymeric Oxygen Sensor
Electron paramagnetic resonance (EPR) oximetry is a powerful technique capable of providing accurate, reliable, and repeated measurements of tissue oxygenation, which is crucial to the diagnosis and treatment of several pathophysiological conditions. Measurement of tissue pO(2) by EPR involves the use of paramagnetic, oxygen-sensitive probes, which can be either soluble (molecular) in nature or insoluble paramagnetic materials. Development of innovative strategies to enhance the biocompatibility and in vivo application of these oxygen-sensing probes is crucial to the growth and clinical applicability of EPR oximetry. Recent research efforts have aimed at encapsulating particulate probes in bioinert polymers for the development of biocompatible EPR probes. In this study, we have developed novel EPR oximetry probes, called perchlorotriphenylmethyl triester (PTM-TE):polydimethyl siloxane (PDMS) chips, by dissolving and incorporating the soluble (molecular) EPR probe, PTM-TE, in an oxygen-permeable polymer matrix, PDMS. We demonstrate that such incorporation (doping) of PTM-TE in PDMS enhanced its oxygen sensitivity several fold. The cast-molding method of fabricating chips enabled them to be made with increasing amounts of PTM-TE (spin density). Characterization of the spin distribution within the PDMS matrix, using EPR micro-imaging, revealed potential inhomogeneties, albeit with no adverse effect on the oxygen-sensing characteristics of PTM-TE:PDMS. The chips were resistant to autoclaving or in vitro oxidoreductant treatment, thus exhibiting excellent in vitro biostability. Our results establish PTM-TE:PDMS as a viable probe for biological oxygen-sensing, and also validate the incorporation of soluble probes in polymer matrices as an innovative approach to the development of novel probes for EPR oximetry.
Molecular Diffusion in Porous Media by PGSE ESR
Diffusion in porous media is a general subject that involves many fields of research, such as chemistry (e.g. porous catalytic pallets), biology (e.g. porous cellular organelles), and materials science (e.g. porous polymer matrixes for controlled-release and gas-storage materials). Pulsed-gradient spin-echo nuclear magnetic resonance (PGSE NMR) is a powerful technique that is often employed to characterize complex diffusion patterns inside porous media. Typically it measures the motion of at least approximately 10(15) molecules occurring in the milliseconds-to-seconds time scale, which can be used to characterize diffusion in porous media with features of approximately 2-3 mum and above (in common aqueous environments). Electron Spin Resonance (ESR), which operates in the nanoseconds-to-microseconds time scale with much better spin sensitivity, can in principle be employed to measure complex diffusion patterns in porous media with much finer features (down to approximately 10 nm). However, up to now, severe technical constraints precluded the adaptation of PGSE ESR to porous media research. In this work we demonstrate for the first time the use of PGSE ESR in the characterization of molecular restricted diffusion in common liquid solutions embedded in a model system for porous media made of sub-micron glass spheres. A unique ESR resonator, efficient gradient coils and fast gradient current drivers enable these measurements. This work can be further extended in the future to many applications that involve dynamical processes occurring in porous media with features in the deep sub-micron range down to true nanometric length scales.
Microimaging of Oxygen Concentration Near Live Photosynthetic Cells by Electron Spin Resonance
We present what is, to our knowledge, a new methodology for high-resolution three-dimensional imaging of oxygen concentration near live cells. The cells are placed in the buffer solution of a stable paramagnetic probe, and electron spin-resonance microimaging is employed to map out the probe's spin-spin relaxation time (T(2)). This information is directly linked to the concentration of the oxygen molecule. The method is demonstrated with a test sample and with a small amount of live photosynthetic cells (cyanobacteria), under conditions of darkness and light. Spatial resolution of approximately 30 x 30 x 100 microm is demonstrated, with approximately microM oxygen concentration sensitivity and sub-fmol absolute oxygen sensitivity per voxel. The use of electron spin-resonance microimaging for oxygen mapping near cells complements the currently available techniques based on microelectrodes or fluorescence/phosphorescence. Furthermore, with the proper paramagnetic probe, it will also be readily applicable for intracellular oxygen microimaging, a capability which other methods find very difficult to achieve.
Sensitive Surface Loop-gap Microresonators for Electron Spin Resonance
This work presents the design, construction, and experimental testing of unique sensitive surface loop-gap microresonators for electron spin resonance (ESR) measurements. These resonators are made of "U"-shaped gold structures with typical sizes of 50 and 150 μm that are deposited on a thin (220 μm) rutile substrate and fed from the rear by a microstrip line. This allows accommodating a large flat sample above the resonator in addition to having variable coupling properties. Such resonators have a very small volume which, compared to previous designs, improves their absolute spin sensitivity by a factor of more than 2 (based on experimental results). They also have a very high microwave field-power conversion ratio of up to 86 gauss/√Hz. This could facilitate the use of very short excitation pulses with relatively low microwave power. Following the presentation and the discussion of the experimental results, ways to further increase sensitivity significantly are outlined.
High-sensitivity Q-band Electron Spin Resonance Imaging System with Submicron Resolution
A pulsed electron spin resonance (ESR) microimaging system operating at the Q-band frequency range is presented. The system includes a pulsed ESR spectrometer, gradient drivers, and a unique high-sensitivity imaging probe. The pulsed gradient drivers are capable of producing peak currents ranging from ∼9 A for short 150 ns pulses up to more than 94 A for long 1400 ns gradient pulses. Under optimal conditions, the imaging probe provides spin sensitivity of ∼1.6 × 10(8) spins∕√Hz or ∼2.7 × 10(6) spins for 1 h of acquisition. This combination of high gradients and high spin sensitivity enables the acquisition of ESR images with a resolution down to ∼440 nm for a high spin concentration solid sample (∼10(8) spins∕μm(3)) and ∼6.7 μm for a low spin concentration liquid sample (∼6 × 10(5) spins/μm(3)). Potential applications of this system range from the imaging of point defects in crystals and semiconductors to measurements of oxygen concentration in biological samples.
Note: High Sensitivity Pulsed Electron Spin Resonance Spectroscopy with Induction Detection
Commercial electron spin resonance spectroscopy and imaging systems make use of the so-called "induction" or "Faraday" detection, which is based on a radio frequency coil or a microwave resonator. The sensitivity of induction detection does not exceed ~3 × 10(8) spins/√Hz. Here we show that through the use of a new type of surface loop-gap microresonators (inner size of 20 μm), operating at cryogenic temperatures at a field of 0.5 T, one can improve upon this sensitivity barrier by more than 2 orders of magnitude and reach spin sensitivities of ~1.5 × 10(6) spins/√Hz or ~2.5 × 10(4) spins for 1 h.
Detection and Imaging of Superoxide in Roots by an Electron Spin Resonance Spin-probe Method
The detection, quantification, and imaging of short-lived reactive oxygen species, such as superoxide, in live biological specimens have always been challenging and controversial. Fluorescence-based methods are nonspecific, and electron spin resonance (ESR) spin-trapping methods require high probe concentrations and lack the capability for sufficient image resolution. In this work, a novel (to our knowledge), sensitive, small ESR imaging resonator was used together with a stable spin probe that specifically reacts with superoxide with a high reaction rate constant. This ESR spin-probe-based methodology was used to examine superoxide generated in a plant root as a result of an apical leaf injury. The results show that the spin probe rapidly permeated the plant's extracellular space. Upon injury of the plant tissue, superoxide was produced and the ESR signal decreased rapidly in the injured parts as well as in the distal part of the root. This is attributed to superoxide production and thus provides a means of quantifying the level of superoxide in the plant. The spin probe's narrow single-line ESR spectrum, together with the sensitive imaging resonator, facilitates the quantitative measurement of superoxide in small biological samples, such as the plant's root, as well as one-dimensional imaging along the length of the root. This type of methodology can be used to resolve many questions involving the production of apoplastic superoxide in plant biology.
