Low-frequency Electron Paramagnetic Resonance Research Articles

Highly Sensitive Detection of Melanin in Melanomas Using Multi-harmonic Low Frequency EPR

PurposeLow frequency EPR can noninvasively detect endogenous free radical melanin in melanocytic skin lesions and could potentially discriminate between benign atypical nevi and malignant melanoma lesions. We recently succeeded in demonstrating the ability of clinical EPR to noninvasively detect the endogenous melanin free radical in skin lesions of patients. However, the signal-to-noise ratio (SNR) was extremely low warranting further research to boost the sensitivity of detection. In the present study, we assessed the performance of a clinical EPR system with the capability to perform multi-harmonic (MH) analysis for the detection of melanin.ProceduresThe sensitivity of MH-EPR was compared with a classical continuous wave (CW)-EPR (1st harmonic) detection in vitro in melanin phantoms, in vivo in melanoma models with cells implanted in the skin, in lymph nodes and having colonized the lungs, and finally on phantoms placed at the surface of human skin.ResultsIn vitro, we observed an increase in SNR by a factor of 10 in flat melanin phantoms when using MH analysis compared to CW combined with an increase in modulation amplitude. In B16 melanomas having grown in the skin of hairless mice, we observed a boost in sensitivity in vivo similar to that observed in vitro with the capability to detect melanoma cells at an earlier stage of development. MH-EPR was also able to detect non-invasively the melanin signal coming from melanoma cells present in lymph nodes as well as in lungs. We also observed a boost of sensitivity using phantoms of melanin placed at the surface of human skin.ConclusionsOverall, our results are paving the way for new clinical trials that will use MH clinical EPR for the characterization of pigmented skin lesions.

Imaging Reactive Oxygen Radicals in Excised Mouse Lung Trapped by Reaction with Hydroxylamine Probes Using 1 GHz Rapid Scan Electron Paramagnetic Resonance.

Oxidative stress is proposed to be critical in acute lung disease, but methods to monitor radicals in lungs are lacking. Our goal is to develop low-frequency electron paramagnetic resonance (EPR) methods to monitor radicals that contribute to the disease. Free radicals generated in a lipopolysaccharide-induced mouse model of acute respiratory distress syndrome reacted with cyclic hydroxylamines CPH (1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine hydrochloride) and DCP-AM-H (4-acetoxymethoxycarbonyl-1-hydroxy-2,2,5,5-tetramethylpyrrolidine-3-carboxylic acid), which were converted into the corresponding nitroxide radicals, CP• and DCP•. The EPR signals of the nitroxide radicals in excised lungs were imaged with a 1 GHz EPR spectrometer/imager that employs rapid scan technology. The small numbers of nitroxides formed by reaction of the hydroxylamine with superoxide result in low signal-to-noise in the spectra and images. However, since the spectral properties of the nitroxides are known, we can use prior knowledge of the line shape and hyperfine splitting to fit the noisy data, yielding well-defined spectra and images. Two-dimensional spectral-spatial images are shown for lung samples containing (4.5 ± 0.5) ×1014 CP• and (9.9 ± 1.0) ×1014 DCP• nitroxide spins. These results suggest that a probe that accumulates in cells gives a stronger nitroxide signal than a probe that is more easily washed out of cells. The nitroxide radicals in excised mouse lungs formed by reaction with hydroxylamine probes CPH and DCP-AM-H can be imaged at 1 GHz.

Broadband EPR Spectroscopy of the Triplet State: Multi-Frequency Analysis of Copper Acetate Monohydrate.

Electron paramagnetic resonance spectroscopy is a long-standing method for the exploration of electronic structures of transition ion complexes. The difficulty of its analysis varies considerably, not only with the nature of the spin system, but more so with the relative magnitudes of the magnetic interactions to which the spin is subject, where particularly challenging cases ensue when two interactions are of comparable magnitude. A case in point is the triplet system S = 1 of coordination complexes with two unpaired electrons when the electronic Zeeman interaction and the electronic zero-field interaction are similar in strength. This situation occurs in the X-band spectra of the thermally excited triplet state of dinuclear copper(II) complexes, exemplified by copper acetate monohydrate. In this study, applicability of the recently developed low-frequency broadband EPR spectrometer to S = 1 systems is investigated on the analysis of multi-frequency, 0.5-16 GHz, data from [Cu(CH3COO)2H2O]2. Global fitting affords the spin Hamiltonian parameters gz = 2.365 ± 0.008; gy = 2.055 ± 0.010; gx = 2.077 ± 0.005; Az = 64 gauss; D = 0.335 ± 0.002 cm-1; E = 0.0105 ± 0.0003 cm-1. The latter two define zero-field absorptions at ca. 630, 7730, and 10,360 MHz, which show up in the spectra as one half of a sharpened symmetrical line. Overall, the EPR line shape is Lorentzian, reflecting spin-lattice relaxation, which is a combination of an unusual, essentially temperature-independent, inverted Orbach process via the S = 0 ground state, and a Raman process proportional to T2. Other broadening mechanisms are limited to at best minor contributions from a distribution in E values, and from dipolar interaction with neighboring copper pairs. Monitoring of a first-order double-quantum transition between 8 and 35 GHz shows a previously unnoticed very complex line shape behavior, which should be the subject of future research.

A Comparative Multi-Frequency EPR Study of Dipolar Interaction in Tetra-Heme Cytochromes.

Distances between Fe ions in multiheme cytochromes are sufficiently short to make the intramolecular dipole-dipole interaction between hemes probable. In the analysis of EPR data from cytochromes, this interaction has thus far been ignored under the assumption that spectra are the simple sum of non-interacting components. Here, we use a recently developed low-frequency broadband EPR spectrometer to establish the extent of dipolar interaction in the example cytochromes, characterize its spectral signatures, and identify present limitations in the analysis. Broadband EPR spectra of Shewanella oneidensis MR-1 small tetraheme cytochrome (STC) have been collected over the frequency range of 0.45 to 13.11 GHz, and they have been compared to similar data from Desulfovibrio vulgaris Hildenborough cytochrome c3. The two cases are representative examples of two very different heme topologies and corresponding electron-transfer properties in tetraheme proteins. While in cytochrome c3, the six Fe-Fe distances can be sorted into two well-separated groups, those in STC are diffuse. Since the onset of dipolar interaction between Fe-Fe pairs is already observed in the X-band, the g values are determined in the simulation of the 13.11 GHz spectrum. Low-frequency spectra are analyzed with the inclusion of dipolar interaction based on available structural data on mutual distances and orientations between all hemes. In this procedure, all 24 possible assignments of individual heme spectra to heme topologies are sampled. The 24 configurations can be reduced to a few, but inspection falls short of a unique assignment, due to a remaining lack of understanding of the fine details of these complex spectra. In general, the EPR analysis suggests the four-heme system in c3 to be more rigid than that in STC, which is proposed to be related to different physiological roles in electron transfer.

Direct Measurement and Imaging of Redox Status with Electron Paramagnetic Resonance.

Significance: Fundamental to the application of tissue redox status to human health is the quantification and localization of tissue redox abnormalities and oxidative stress and their correlation with the severity and local extent of disease to inform therapy. The centrality of the low-molecular-weight thiol, glutathione, in physiological redox balance has long been appreciated, but direct measurement of tissue thiol status in vivo has not been possible hitherto. Recent advances in instrumentation and molecular probes suggest the feasibility of real-time redox assessment in humans. Recent Advances: Recent studies have demonstrated the feasibility of using low-frequency electron paramagnetic resonance (EPR) techniques for quantitative imaging of redox status in mammalian tissues in vivo. Rapid-scan (RS) EPR spectroscopy and imaging, new disulfide-dinitroxide spin probes, and novel analytic techniques have led to significant advances in direct, quantitative imaging of thiol redox status. Critical Issues: While novel RS EPR imaging coupled with first-generation molecular probes has demonstrated the feasibility of imaging thiol redox status in vivo, further technical advancements are desirable and ongoing. These include developing spin probes that are tailored for specific tissues with response kinetics tuned to the physiological environment. Equally critical are RS instrumentation with higher signal-to-noise ratio and minimal signal distortion, as well as optimized imaging protocols for image acquisition with sparsity adapted to image information content. Future Directions: Quantitative images of tissue glutathione promise to enable acquisition of a general image of mammalian and potentially human tissue health.

Statins Alleviate Tumor Hypoxia in Prostate Cancer Models by Decreasing Oxygen Consumption: An Opportunity for Radiosensitization?

Background: Because statins were found to decrease the oxygen consumption rate (OCR) of a variety of normal cells, our hypothesis was that statins may also decrease the OCR of cancer cells, alleviate tumor hypoxia and radiosensitize tumors. Methods: OCR was assessed using the Seahorse XF96 technology and EPR respirometry in PC-3 prostate cancer cells. Mitochondrial superoxide production was measured by EPR with mitoTEMPO-H as a sensing probe. Tumor pO2 was measured in vivo using low-frequency EPR oximetry to define the optimal window of reoxygenation, the time at which tumors were irradiated with a single 6 Gy dose with a Cesium-137 irradiator. Results: 24-h exposure to simvastatin and fluvastatin significantly decreased the OCR of PC-3 cancer cells. An increase in mitochondrial superoxide levels was also observed after fluvastatin exposure. The PC-3 prostate cancer model was found highly hypoxic at the basal level. When mice were treated with simvastatin or fluvastatin (daily injection of 20 mg/kg), tumor oxygenation increased 48 and 72 h after initiation of the treatment. However, despite reoxygenation, simvastatin did not sensitize the PC-3 tumor model to RT. Conclusions: exposure to statins affect tumor metabolism and tumor oxygenation, however, with limited impact on tumor growth with or without irradiation.

Noninvasive detection of the endogenous free radical melanin in human skin melanomas using electron paramagnetic resonance (EPR).

We explored the capability of low-frequency Electron Paramagnetic Resonance (EPR) to noninvasively detect melanin (a stable semiquinone free radical) in the human skin. As previous in vitro studies on biopsies suggested that the EPR signal from melanin was different when measured in skin melanomas or benign nevi, we conducted a prospective first-in-man clinical EPR study in patients with skin lesions suspicious of melanoma. EPR spectra were obtained using a spectrometer operating at 1GHz, with a surface coil placed over the area of interest. Two clinical studies were carried out: 1) healthy volunteers (n=45) presenting different skin phototypes; 2) patients (n=88) with skin lesions suspicious of melanoma (n=100) requiring surgical resection. EPR data obtained before surgery were compared with histopathology results. The method was not sensitive enough to measure differences in melanin content due to changes in skin pigmentation. In patients, 92% of the spectra were analyzable. The EPR signal of melanin was significantly higher (p<0.0001) in melanoma lesions (n=26) than that in benign atypical nevi (n=62). A trend toward a higher signal intensity (though not significant) was observed in high Breslow depth melanomas (a marker of skin invasion) than in low Breslow lesions. To date, no naturally occurring free radicals have been detected by low-frequency EPR systems adapted for clinical studies. Here, we demonstrated for the first time the ability of this technology to detect an endogenous free radical, opening new avenues for evaluating clinical EPR as a potential aid in the diagnosis of pigmented skin lesions.

Low-frequency EPR of ferrimyoglobin fluoride and ferrimyoglobin cyanide: a case study on the applicability of broadband analysis to high-spin hemoproteins and to HALS hemoproteins

An EPR spectrometer has been developed that can be tuned to many frequencies in the range of ca 0.1–15 GHz. Applicability has been tested on ferrimyoglobin fluoride (MbF) and ferrimyoglobin cyanide (MbCN). MbF has a high-spin (S = 5/2) spectrum with 19F superhyperfine splitting that is only resolved in X-band along the heme normal. Low-frequency EPR also resolves the splitting in the heme plane. Measurement of linewidth as a function of frequency provides the basis for an analysis of inhomogeneous broadening in terms of g-strain, zero-field distribution, unresolved superhyperfine splittings and dipolar interaction. Rhombicity in the g tensor is found to be absent. MbCN (S = 1/2) has a highly anisotropic low spin (HALS) spectrum for which gx cannot be determined unequivocally in X-band. Low-frequency EPR allows for measurement of the complete spectrum and determination of the g-tensor.Graphical abstract

A Review of Low-Frequency EPR Technology for the Measurement of Brain pO2 and Oxidative Stress.

EPR can uniquely measure paramagnetic species. Although commercial EPR was introduced in 1950s, the early studies were mostly restricted to chemicals in solution or cellular experiments using X-band EPR equipment. Due to its limited penetration (<1 mm), experiments with living animals were almost impossible. To overcome these difficulties, Swartz group, along with several other leaders in field, pioneered the technology of low frequency EPR (e.g., L-band, 1–2 GHz). The development of low frequency EPR and the associated probes have dramatically expanded the application of EPR technology into the biomedical research field, providing answers to important scientific questions by measuring specific parameters that are impossible or very difficult to obtain by other approaches. In this review, which is aimed at highlighting the seminal contribution from Swartz group over the last several decades, we will focus on the development of EPR technology that was designed to deal with the potential challenges arising from conducting EPR spectroscopy in living animals. The second half of the review will be concentrated on the application of low frequency EPR in measuring cerebral tissue pO2 changes and oxidative stress in various physiological and pathophysiological conditions in the brain of animal disease models.

Mapping Pigments in a Painting with Low Frequency Electron Paramagnetic Resonance Spectroscopy

An electron paramagnetic resonance (EPR) mobile universal surface explorer (MOUSE) was recently introduced for noninvasively studying paramagnetic pigments in paintings. This study determined that the EPR MOUSE could map the spatial locations of four pigments in a simple impasto painting. Results from three spectral identification algorithms were examined to assess their ability to identify the pigments using an unsupervised approach. Resulting pigment maps are displayed as colorized images of the spatial distribution of the pigments. All three algorithms produced reasonable representations of the painting. The algorithms achieved excellent true positive, true negative, false positive, and false negative rates of ≥0.95, ≥0.98, ≤0.02, and ≤0.05, respectively, for the identification of the pigments. We conclude that the EPR MOUSE is suitable for accurately mapping the location of paramagnetic pigments in a painting.

Ligand control of low-frequency electron paramagnetic resonance linewidth in Cr(III) complexes.

Understanding how the ligand shell controls low-frequency electron paramagnetic resonance (EPR) spectroscopic properties of metal ions is essential if they are to be used in EPR-based bioimaging schemes. In this work, we probe how specific variations in the ligand structure impact L-band (ca. 1.3 GHz) EPR spectroscopic linewidths in the trichloride salts of five Cr(iii) complexes: [Cr(RR-dphen)3]3+ (RR-dphen = (1R,2R)-(+)-diphenylethylenediamine, 1), [Cr(en)3]3+ (en = ethylenediamine, 2), [Cr(me-en)3]3+ (me-en = 1,2-diaminopropane, 3), [Cr(tn)3]3+ (tn = 1,3-diaminopropane, 4) [Cr(trans-chxn)3]3+ (trans-chxn = trans-(±)-1,2-diaminocyclohexane, 5). Spectral broadening varies in a nonintuitive manner across the series, showing the sharpest peaks for 1 and broadest for 5. Molecular dynamics simulations provide evidence that the broadening is correlated to rigidity in the inner coordination sphere and reflected in ligand-dependent distribution of Cr-N bond distances that can be found in frozen solution.

Resolved Hyperfine at L-band for High-Spin CoEDTA, A Model for Co Sites in Proteins.

Low-frequency electron paramagnetic resonance (EPR) spectra were obtained for the Co complex of ethylene diamine tetraacetic acid (CoEDTA). It was found that the cobalt hyperfine at geff-mid is better resolved at a low frequency, L-band (1.37 GHz), and not resolved at X-band (9.631 GHz), which is the conventional frequency used for most spectra for metal complexes. Resolved cobalt hyperfine lines lead to additional EPR parameters like A-mid for cobalt and a more-accurate determination of g-mid. Resolved hyperfine lines in the L-band, but not the S-band, spectra were obtained at a concentration of 1 mM. Knowing these additional EPR parameters provides a means to better determine the electron density in the ground state orbital for each cobalt complex, as well as to determine differences upon a change of ligation. If zinc sites can be replaced by cobalt, the cobalt spectra for these sites will enhance the characterization of the zinc sites.

Better Resolution of High-Spin Cobalt Hyperfine at Low Frequency: Co-Doped Ba(Zn1/3Ta2/3)O₃ as a Model Complex.

Low-frequency electron paramagnetic resonance (EPR) is used to extract the EPR parameter A-mid and support the approximate X-band value of g-mid for Ba(CoyZn1/3−yTa2/3)O3. Although the cobalt hyperfine structure for the |±1/2〉 state is often unresolved at X-band or S-band, it is resolved in measurements on this compound. This allows for detailed analysis of the molecular orbital for the |±1/2〉 state, which is often the ground state. Moreover, this work shows that the EPR parameters for Co substituted into Zn compounds give important insight into the properties of zinc binding sites.

Characterization of a clinically used charcoal suspension for in vivo EPR oximetry.

Electron paramagnetic resonance (EPR) oximetry using particulate materials allows repeatable measurements of oxygen in tissues. However, the materials identified so far are not medical devices, thus precluding their immediate use in clinical studies. The aim of this study was to assess the magnetic properties of Carbo-Rep®, a charcoal suspension used as a liquid marker for preoperative tumor localization. Calibration curves (EPR linewidth as a function of pO2) were built using 9-GHz EPR spectrometry. The feasibility of performing oxygen measurements was examined in vivo by using a low-frequency (1GHz) EPR spectrometer and by inducing ischemia in the gastrocnemius muscle of mice or by submitting rats bearing tumors to different oxygen-breathing challenges. Paramagnetic centers presenting a high oxygen sensitivity were identified in Carbo-Rep®. At 1GHz, the EPR linewidth varied from 98 to 426µT in L-band in nitrogen and air, respectively. The sensor allowed repeated measurements of oxygen over 6months in muscles of mice. Subtle variations of tumor oxygenation were monitored in rats when switching gas breathing from air to carbogen. The magnetic properties of Carbo-Rep® are promising for its future use as oxygen sensor in clinical EPR oximetry.

A nondestructive method of identifying pigments on canvas using low frequency electron paramagnetic resonance spectroscopy

ABSTRACTElectron paramagnetic resonance (EPR) spectroscopy is a useful analytical tool for studying objects with cultural heritage significance. It is generally thought of as a destructive spectroscopy, owing to the small sample size, and thus not used to study paintings. We demonstrate that a form of this spectroscopy known as low frequency EPR spectroscopy can be used to nondestructively identify ultramarine blue, Egyptian blue, Han blue, rhodochrosite, blue vitriol, terracotta red, charcoal, and coal pigments in a linseed oil paint on canvas.

Tabletop 700 MHz electron paramagnetic resonance imaging spectrometer.

Low frequency electron paramagnetic resonance imaging is a powerful tool to non-invasively measure the physiological status of tumors. Here, we report on the design and functionality of a rapid scan and pulse table-top imaging spectrometer based around an arbitrary waveform generator and 25mm cross-loop resonator operating at 700 MHz. Two and four-dimensional rapid scan spectral-spatial images are presented. This table-top imager is a prototype for future pre-clinical imagers.

Low-frequency ESR studies on permeable and impermeable deuterated nitroxyl radicals in corn oil solution.

Low-frequency electron spin resonance studies were performed for 2mM concentration of deuterated permeable and impermeable nitroxyl spin probes, 3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine-1-oxyl and 3-carboxy-2,2,5,5,-tetramethyl-1-pyrrolidinyloxy in pure water and various concentrations of corn oil solution. The electron spin resonance parameters such as the line width, hyperfine coupling constant, g factor, rotational correlation time, permeability, and partition parameter were estimated. The broadening of line width was observed for nitroxyl radicals in corn oil mixture. The rotational correlation time increases with increasing concentration of corn oil, which indicates the less mobile nature of spin probe in corn oil mixture. The membrane permeability and partition parameter values were estimated as a function of corn oil concentration, which reveals that the nitroxyl radicals permeate equally into the aqueous phase and oil phase at the corn oil concentration of 50%. The electron spin resonance spectra demonstrate the permeable and impermeable nature of nitroxyl spin probes. From these results, the corn oil concentration was optimized as 50% for phantom studies. In this work, the corn oil and pure water mixture phantom models with various viscosities correspond to plasma membrane, and whole blood membrane with different hematocrit levels was studied for monitoring the biological characteristics and their interactions with permeable nitroxyl spin probe. These results will be useful for the development of electron spin resonance and Overhauser-enhanced magnetic resonance imaging modalities in biomedical applications.

An electron paramagnetic resonance mobile universal surface explorer

Many samples cannot be studied by electron paramagnetic resonance (EPR) spectroscopy because they are too large to fit into the spectrometer and too precious to be destructively sampled for study. An EPR mobile universal surface explorer (MOUSE), also known as a unilateral EPR spectrometer, was constructed for studying this class of sample. The EPR MOUSE can nondestructively record a low frequency EPR (LFEPR) spectrum of a small region of any size object by placing the MOUSE against the object. The capabilities of the EPR MOUSE are demonstrated on paramagnetic paint pigments on canvas, magnetic ink on paper, and a ceramic candlestick. The mobile nature of the MOUSE will allow the spectrometer to be brought to the sample, thus opening new applications of EPR spectroscopy.

EPR monitoring of wound oxygenation as a biomarker of response to gene therapy encoding hCAP-18/LL37 peptide.

To investigate the value of electron paramagnetic resonance oximetry to follow oxygenation in wounds treated by a plasmid-encoding host defense peptide hCAP-18/LL37. Flaps were created on diabetic mice (7- or 12-week-old db/db mice) presenting different levels of microangiopathy. The hCAP-18/LL37-encoding plasmids were administered in wounds by electroporation. Low-frequency electron paramagnetic resonance oximetry using lithium phthalocyanine as the oxygen sensor was used to monitor wound oxygenation in flaps during the healing process. Flaps were analyzed by immunohistochemistry to assess hypoxia and cell proliferation. Kinetics of closure was also assessed in excisional skin wounds. A reoxygenation of the flap was observed during the healing process in the 7-week-old db/db treated mice, but not in the untreated mice and the 12-week-old mice. Histological studies demonstrated less hypoxic regions and higher proportion of proliferating cells in hCAP-18/LL37-treated flaps in the 7-week-old db/db treated mice compared with untreated mice. Consistently, the kinetics of excisional wound closure was improved by hCAP-18/LL37 treatment in the 7-week-old db/db but not in the 12-week-old mice. Oxygenation measured by electron paramagnetic resonance oximetry is a promising biomarker of response to treatments designed to modulate wound oxygenation. Magn Reson Med 79:3267-3273, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

Rapid-scan EPR imaging

In rapid-scan EPR the magnetic field or frequency is repeatedly scanned through the spectrum at rates that are much faster than in conventional continuous wave EPR. The signal is directly-detected with a mixer at the source frequency. Rapid-scan EPR is particularly advantageous when the scan rate through resonance is fast relative to electron spin relaxation rates. In such scans, there may be oscillations on the trailing edge of the spectrum. These oscillations can be removed by mathematical deconvolution to recover the slow-scan absorption spectrum. In cases of inhomogeneous broadening, the oscillations may interfere destructively to the extent that they are not visible. The deconvolution can be used even when it is not required, so spectra can be obtained in which some portions of the spectrum are in the rapid-scan regime and some are not. The technology developed for rapid-scan EPR can be applied generally so long as spectra are obtained in the linear response region. The detection of the full spectrum in each scan, the ability to use higher microwave power without saturation, and the noise filtering inherent in coherent averaging results in substantial improvement in signal-to-noise relative to conventional continuous wave spectroscopy, which is particularly advantageous for low-frequency EPR imaging. This overview describes the principles of rapid-scan EPR and the hardware used to generate the spectra. Examples are provided of its application to imaging of nitroxide radicals, diradicals, and spin-trapped radicals at a Larmor frequency of ca. 250MHz.