Rapid Scan Electron Paramagnetic Resonance Research Articles

Field-domain rapid-scan EPR at 240 GHz for studies of protein functional dynamics at room temperature

We present field-domain rapid-scan (RS) electron paramagnetic resonance (EPR) at 8.6T and 240GHz. To enable this technique, we upgraded a home-built EPR spectrometer with an FPGA-enabled digitizer and real-time processing software. The software leverages the Hilbert transform to recover the in-phase (I) and quadrature (Q) channels, and therefore the raw absorptive and dispersive signals, χ′ and χ′′, from their combined magnitude (I2+Q2). Averaging a magnitude is simpler than real-time coherent averaging and has the added benefit of permitting long-timescale signal averaging (up to at least 2.5×106 scans) because it eliminates the effects of source-receiver phase drift. Our rapid-scan (RS) EPR provides a signal-to-noise ratio that is approximately twice that of continuous wave (CW) EPR under the same experimental conditions, after scaling by the square root of acquisition time. We apply our RS EPR as an extension of the recently reported time-resolved Gd-Gd EPR (TiGGER) [Maity et al., 2023], which is able to monitor inter-residue distance changes during the photocycle of a photoresponsive protein through changes in the Gd-Gd dipolar couplings. RS, opposed to CW, returns field-swept spectra as a function of time with 10ms time resolution, and thus, adds a second dimension to the static field transients recorded by TiGGER. We were able to use RS TiGGER to track time-dependent and temperature-dependent kinetics of AsLOV2, a light-activated phototropin domain found in oats. The results presented here combine the benefits of RS EPR with the improved spectral resolution and sensitivity of Gd chelates at high magnetic fields. In the future, field-domain RS EPR at high magnetic fields may enable studies of other real-time kinetic processes with time resolutions that are otherwise difficult to access in the solution state.

Real-Time Monitoring of Photoinduced pH Jumps by In Situ Rapid-Scan EPR Spectroscopy.

This work represents the first demonstration of monitoring kinetics upon a light-induced pH jump by in situ rapid-scan (RS) electron paramagnetic resonance (EPR) spectroscopy on the millisecond time scale. Here, we focus on the protonation state of an imidazolidine type radical as a pH sensor under visible light irradiation of a merocyanine photoacid in bulk solution. The results highlight the utility of photoacids in combination with pH-sensitive spin probes as an effective tool for the real-time investigation of biochemical mechanisms regulated by changes in the pH value.

Electron Spin Resonance Probe Incorporation into Bioinks Permits Longitudinal Oxygen Imaging of Bioprinted Constructs

PurposeBioprinting is an additive manufacturing technology analogous to 3D printing. Instead of plastic or resin, cell-laden hydrogels are used to produce a construct of the intended biological structure. Over time, cells transform this construct into a functioning tissue or organ. The process of printing followed by tissue maturation is referred to as 4D bioprinting. The fourth dimension is temporal. Failure to provide living cells with sufficient amounts of oxygen at any point along the developmental timeline may jeopardize the bioprinting goals. Even transient hypoxia may alter cells' differentiation and proliferation or trigger apoptosis. Electron paramagnetic resonance (EPR) imaging modality is proposed to permit 4D monitoring of oxygen within bioprinted structures.ProceduresLithium octa-n-butoxy-phthalocyanine (LiNc-BuO) probes have been introduced into gelatin methacrylate (GelMA) bioink. GelMA is a cross-linkable hydrogel, and LiNc-BuO is an oxygen-sensitive compound that permits longitudinal oximetric measurements. The effects of the oxygen probe on printability have been evaluated. A digital light processing (DLP) bioprinter was built in the laboratory. Bioprinting protocols have been developed that consider the optical properties of the GelMA/LiNc-BuO composites. Acellular and cell-laden constructs have been printed and imaged. The post-printing effect of residual photoinitiator on oxygen depletion has been investigated.ResultsModels have been successfully printed using a lab-built bioprinter. Rapid scan EPR images reflective of the expected oxygen concentration levels have been acquired. An unreported problem of oxygen depletion in bioprinted constructs by the residual photoinitiator has been documented. EPR imaging is proposed as a control method for its removal. The oxygen consumption rates by HEK293T cells within a bioprinted cylinder have been imaged and quantified.ConclusionsThe feasibility of the cointegration of 4D EPR imaging and 4D bioprinting has been demonstrated. The proof-of-concept experiments, which were conducted using oxygen probes loaded into GelMA, lay the foundation for a broad range of applications, such as bioprinting with many types of bioinks loaded with diverse varieties of molecular spin probes.

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.

Background removal from rapid-scan EPR spectra of nitroxide-based spin labels by minimizing non-quadratic cost functions

Rapid-scan electron paramagnetic resonance spectroscopy is an emerging technique which substantially improves the signal-to-noise ratio and time resolution compared to conventional continuous-wave experiments. This allows the investigation of spin-labeled biomolecules and their structural dynamics on much shorter time scales than usually accessible. The EPR spectrum however is superimposed by a strong background that is caused by microphonic effects of the alternating magnetic field. This article discusses the use of non-quadratic cost functions for background removal of rapid-scan spectra. The method is validated for the most prominent type of spin-probes in the field of biochemistry: the nitroxide spin-label.

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.

Rapid Scan Electron Paramagnetic Resonance Spectroscopy Is a Suitable Tool to Study Intermolecular Interactions of Intrinsically Disordered Protein.

Intrinsically disordered proteins (IDPs) are involved in most crucial cellular processes. However, they lack a well-defined fold hampering the investigation of their structural ensemble and interactions. Suitable biophysical methods able to manage their inherent flexibility and broad conformational ensemble are scarce. Here, we used rapid scan (RS) electron paramagnetic resonance (EPR) spectroscopy to study the intermolecular interactions of the IDP α-synuclein (aS). aS aggregation and fibril deposition is the hallmark of Parkinson's disease, and specific point mutations, among them A30P and A53T, were linked to the early onset of the disease. To understand the pathological processes, research intensively investigates aS aggregation kinetics, which was reported to be accelerated in the presence of ethanol. Conventional techniques fail to capture these fast processes due to their limited time resolution and, thus, lose kinetic information. We have demonstrated that RS EPR spectroscopy is suitable for studying aS aggregation by resolving underlying kinetics and highlighting differences in fibrillization behavior. RS EPR spectroscopy outperforms traditional EPR methods in terms of sensitivity by a factor of 5 in our case while significantly reducing data acquisition time. Thus, we were able to sample short time intervals capturing single events taking place during the aggregation process. Further studies will therefore be able to shed light on biological processes proceeding on fast time scales.

Rapid scan EPR: Automated digital resonator control for low-latency data acquisition

Automation has become an essential component of modern scientific instruments which often capture large amounts of complex dynamic data. Algorithms are developed to read multiple sensors in parallel with data acquisition and to adjust instrumental parameters on the fly. Decisions are made on a time scale unattainable to the human operator. In addition to speed, automation reduces human error, improves the reproducibility of experiments, and improves the reliability of acquired data. An automatic digital control (ADiC) was developed to reliably sustain critical coupling of a resonator over a wide range of time-varying loading conditions. The ADiC uses the computational power of a microcontroller that directly communicates with all system components independent of a personal computer (PC). The PC initiates resonator tuning and coupling by sending a command to MC via serial port. After receiving the command, ADiC establishes critical coupling conditions within approximately 5 ms. A printed circuit board resonator was designed to permit digital control. The performance of the resonator together with the ADiC was evaluated by varying the resonator loading from empty to heavily loaded. For the loading, samples containing aqueous sodium chloride that strongly absorb electromagnetic waves were used. A previously reported rapid scan (RS) electron paramagnetic resonance (EPR) imaging instrument was upgraded by the incorporation of ADiC. RS spectra and an in vivo image of oxygen in a mouse tumor model have been acquired using the upgraded system. ADiC robustly sustained critical coupling of the resonator to the transmission line during these measurements. The design implemented in this study can be used in slow-scan and pulsed EPR with modifications.

A computationally efficient discrete pseudomodulation algorithm for real-time magnetic resonance measurements.

Rapid-scan electron paramagnetic resonance (RSEPR) results in a significant improvement in signal-to-noise over magnetic field modulated continuous wave EPR (CWEPR). However, the RSEPR raw absorption spectra can make the real-time comparison of CWEPR spectra difficult, especially in systems where the total number of paramagnetic spins is low. In this paper, we illustrate a method of applying pseudomodulation within RSEPR data collection software in real-time. Pseudomodulation is generally carried out in post-processing to increase signal-to-noise and simulate the effects of modulation on the spectra observed in traditional magnetic field modulated CWEPR. By applying the pseudomodulation method on a discrete computational basis, the technique can be utilized in parallel with data collection due to the significantly reduced computational power of the discretized pseudomodulation calculation. This allows for the live alteration of modulation parameters, such as the modulation amplitude and modulation harmonic. This real-time simulation allows for the comparison of the accumulated non-adiabatic rapid-sweep EPR spectra with the known CWEPR spectra available in the literature and has the ability to view smaller and less sensitive resonance features for various harmonics during high-frequency experiments while retaining all signal-to-noise improvements.

Rapid Scan EPR Oxygen Imaging in Photoactivated Resin Used for Stereolithographic 3D Printing.

Oxygen plays a critical role in the photopolymerization process resulting in the formation of solid structures from liquid resins during three-dimensional (3D) printing: it acts as a polymerization inhibitor. Upon exposure to light, oxygen is depleted. As a result, the polymerization process becomes activated. Electron paramagnetic resonance (EPR) imaging is described as a tool to visualize changes in oxygen distribution caused by light exposure. This nondestructive method uses radio waves and, therefore, is not constrained by optical opacity offering greater penetrating depth. Three proof-of-principle imaging experiments were demonstrated: (1) spatial propagation of the photopolymerization process; (2) oxygen depletion as a result of postcuring; and (3) oxygen visualization in a 3D printed spiral model. Commercial stereolithography (SLA) resin was used in these experiments. Lithium octa-n-butoxynaphthalocyanine (LiNc-BuO) probe was mixed with the resin to permit oxygen imaging. Li-naphthalocyanine probes are routinely used in various EPR applications because of their long-term stability and high functional sensitivity to oxygen. In this study, we demonstrate that EPR imaging has the potential to become a powerful visualization tool in the development of 3D printing technology, including bioprinting and tissue engineering.

Development of an L-band resonator optimized for fast scan EPR imaging of the mouse head.

To develop a novel resonator for high-quality fast scan electron paramagnetic resonance (EPR) and EPR/NMR co-imaging of the head and brain of mice at 1.25 GHz. Resonator dimensions were scaled to fit the mouse head with maximum filling factor. A single-loop 6-gap resonator of 20 mm diameter and 20 mm length was constructed. High resonator stability was achieved utilizing a fixed position double coupling loop. Symmetrical mutually inverted connections rendered it insensitive to field modulation and fast scan. Coupling adjustment was provided by a parallel-connected variable capacitor located at the feeding line at λ/4 distance. To minimize radiation loss, the shield around the resonator was supplemented with a planar conductive disc that focuses return magnetic flux. Coupling of the resonator loaded with the mouse head was efficient and easy. This resonator enabled high-quality in vivo 3D EPR imaging of the mouse head following intravenous infusion of nitroxide probes. With this resonator and rapid scan EPR system, 4 ms scans were acquired in forward and reverse directions so that images with 2-scan 3,136 projections were acquired in 25 s. Head images were achieved with resolutions of 0.4 mm, enabling visualization of probe localization and uptake across the blood-brain barrier. This resonator design provides good sensitivity, high stability, and B1 field homogeneity for in vivo fast scan EPR of the mouse head and brain, enabling faster measurements and higher resolution imaging of probe uptake, localization, and metabolism than previously possible.

Intracellular Protein–Lipid Interactions Studied by Rapid-Scan Electron Paramagnetic Resonance Spectroscopy

Protein–membrane interactions play key roles in essential cellular processes; studying these interactions in the cell is a challenging task of modern biophysical chemistry. A prominent example is the interaction of human α-synuclein (αS) with negatively charged membranes. It has been well-studied in vitro, but in spite of the huge amount of lipid membranes in the crowded environment of biological cells, to date, no interactions have been detected in cells. Here, we use rapid-scan (RS) electron paramagnetic resonance (EPR) spectroscopy to study αS interactions with negatively charged vesicles in vitro and upon transfection of the protein and lipid vesicles into model cells, i.e., oocytes of Xenopus laevis. We show that protein–vesicle interactions are reflected in RS spectra in vitro and in cells, which enables time-resolved monitoring of protein–membrane interaction upon transfection into cells. Our data suggest binding of a small fraction of αS to endogenous membranes.

Rapid‐Scan Electron Paramagnetic Resonance of Highly Resolved Hyperfine Lines in Organic Radicals.

X-band (ca. 9 GHz) fluid solution rapid-scan electron paramagnetic resonance spectra are reported for radicals with multiline spectra and resolution of hyperfine lines as narrow as 30 mG. Highly-resolved spectra of 3-carbamoyl-2,2,5,5-tetramethylpyrrolidin-1-yloxy, diphenylnitroxide, galvinoxyl, and perylene cation radical with excellent signal-to-noise are shown, demonstrating the capabilities of the rapid-scan technique to characterize very small, well-resolved hyperfine couplings. To acquire high resolution spectra the signal bandwidth must be less than the resonator bandwidth. Signal bandwidth is inversely proportional to linewidth and proportional to scan rate. Resonator bandwidth is inversely proportional to resonator Q. Proper selection of scan rate and resonator Q is needed to achieve resolution of closely-spaced narrow EPR lines.

Modular imaging system: Rapid scan EPR at 800 MHz.

An electron paramagnetic resonance (EPR) imaging system has been custom built for use in pre-clinical and, potentially, clinical studies. Commercial standalone modules have been used in the design that are MATLAB-controlled. The imaging system combines digital and analog technologies. It was designed to achieve maximum flexibility and versatility and to perform standard and novel user-defined experiments. This design goal is achieved by frequency mixing of an arbitrary waveform generator (AWG) output at the intermediate frequency (IF) with a constant source frequency (SF). Low noise SF at 250, 750, and 1000 MHz are available in the system. A wide range of frequencies from near-baseband to L-band can be generated as a result. Two-stage downconversion at the signal detection side is implemented that enables multi-frequency EPR capability. In the first stage, the signal frequency is converted to IF. A novel AWG-enabled digital auto-frequency control method that operates at IF is described that is used for automatic resonator tuning. Quadrature baseband EPR signal is generated in the second downconversion step. The semi-digital approach of mixing low-noise frequency sources with an AWG permits generation of arbitrary excitation patterns that include but are not limited to frequency sweeps for resonator tuning and matching, continuous-wave, and pulse sequences. Presented in this paper is the demonstration of rapid scan (RS) EPR imaging implemented at 800 MHz. Generation of stable magnetic scan waveforms is critical for the RS method. A digital automatic scan control (DASC) system was developed for sinusoidal magnetic field scans. DASC permits tight control of both amplitude and phase of the scans. A surface loop resonator was developed using 3D printing technology. RS EPR imaging system was validated using sample phantoms. In vivo imaging of a breast cancer mouse model is demonstrated.

Development of an L-band rapid scan EPR digital console.

The development of a digital console for in-vivo rapid scan electron paramagnetic resonance (RS-EPR) spectroscopy and imaging is described in detail. The console was build using field programmable gate array (FGPA) technology that permits real-time control of the resonator and scanning magnetic fields during the measurements. Automatic resonator tuning and matching are achieved by implementing a digital feedback control system and using voltage-tunable capacitors. A band-pass subsampling method is used to directly digitize EPR signals at the carrier frequencies of about 1.2 GHz. The magnetic field scan waveforms, excitation EPR frequency, and sampling clock are all internally synchronized. Full-cycle RS-EPR signals are accumulated in the FPGA in real time without any time gaps. The result is the elimination of the re-arm time, during which data are not acquired. The proposed design in this manuscript has a small footprint and is relatively low cost. The FPGA-based RS-EPR system was tested using standard LiNc-BuO and tempone-d16 samples. The RS-EPR linewidth of the LiNc-BuO sample was consistent with an independent pulsed EPR measurement.

250 MHz Rapid Scan Cross Loop Resonator.

A 25 mm diameter 250 MHz crossed-loop resonator was designed for rapid scan electron paramagnetic resonance imaging. It has a saddle coil for the driven resonator and a fine wire, loop gap resonator for the sample resonator. There is good separation of E and B fields and high isolation between the two resonators, permitting a wide range of sample types to be measured. Applications to imaging of nitroxide, trityl, and LiPc samples illustrate the utility of the resonator. Using this resonator and a trityl sample the signal-to-noise of a rapid scan absorption spectrum is about 20 times higher than for a first-derivative CW spectrum.

Full cycle rapid scan EPR deconvolution algorithm

Rapid scan electron paramagnetic resonance (RS EPR) is a continuous-wave (CW) method that combines narrowband excitation and broadband detection. Sinusoidal magnetic field scans that span the entire EPR spectrum cause electron spin excitations twice during the scan period. Periodic transient RS signals are digitized and time-averaged. Deconvolution of absorption spectrum from the measured full-cycle signal is an ill-posed problem that does not have a stable solution because the magnetic field passes the same EPR line twice per sinusoidal scan during up- and down-field passages. As a result, RS signals consist of two contributions that need to be separated and postprocessed individually. Deconvolution of either of the contributions is a well-posed problem that has a stable solution. The current version of the RS EPR algorithm solves the separation problem by cutting the full-scan signal into two half-period pieces. This imposes a constraint on the experiment; the EPR signal must completely decay by the end of each half-scan in order to not be truncated. The constraint limits the maximum scan frequency and, therefore, the RS signal-to-noise gain. Faster scans permit the use of higher excitation powers without saturating the spin system, translating into a higher EPR sensitivity. A stable, full-scan algorithm is described in this paper that does not require truncation of the periodic response. This algorithm utilizes the additive property of linear systems: the response to a sum of two inputs is equal the sum of responses to each of the inputs separately. Based on this property, the mathematical model for CW RS EPR can be replaced by that of a sum of two independent full-cycle pulsed field-modulated experiments. In each of these experiments, the excitation power equals to zero during either up- or down-field scan. The full-cycle algorithm permits approaching the upper theoretical scan frequency limit; the transient spin system response must decay within the scan period. Separation of the interfering up- and down-field scan responses remains a challenge for reaching the full potential of this new method. For this reason, only a factor of two increase in the scan rate was achieved, in comparison with the standard half-scan RS EPR algorithm. It is important for practical use that faster scans not necessarily increase the signal bandwidth because acceleration of the Larmor frequency driven by the changing magnetic field changes its sign after passing the inflection points on the scan. The half-scan and full-scan algorithms are compared using a LiNC-BuO spin probe of known line-shape, demonstrating that the new method produces stable solutions when RS signals do not completely decay by the end of each half-scan.

Imaging thiol redox status in murine tumors in vivo with rapid-scan electron paramagnetic resonance

Thiol redox status is an important physiologic parameter that affects the success or failure of cancer treatment. Rapid scan electron paramagnetic resonance (RS EPR) is a novel technique that has shown higher signal-to-noise ratio than conventional continuous-wave EPR in in vitro studies. Here we used RS EPR to acquire rapid three-dimensional images of the thiol redox status of tumors in living mice. This work presents, for the first time, in vivo RS EPR images of the kinetics of the reaction of 2H,15N-substituted disulfide-linked dinitroxide (PxSSPx) spin probe with intracellular glutathione. The cleavage rate is proportional to the intracellular glutathione concentration. Feasibility was demonstrated in a FSa fibrosarcoma tumor model in C3H mice. Similar to other in vivo and cell model studies, decreasing intracellular glutathione concentration by treating mice with l-buthionine sulfoximine (BSO) markedly altered the kinetic images.

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters <em>In Vivo</em>

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan electron-paramagnetic-resonance (RS-EPR), which can provide quantitative information under in vivo conditions on oxygen concentration, pH, redox status and concentration of signaling molecules (i.e., OH•, NO•). The RS-EPR technique has a higher sensitivity, improved spatial resolution (1 mm), and shorter acquisition time in comparison to the standard continuous wave (CW) technique. A variety of phantom configurations have been tested, with spatial resolution varying from 1 to 6 mm, and spectral width of the reporter molecules ranging from 16 µT (160 mG) to 5 mT (50 G). A cross-loop bimodal resonator decouples excitation and detection, reducing the noise, while the rapid scan effect allows more power to be input to the spin system before saturation, increasing the EPR signal. This leads to a substantially higher signal-to-noise ratio than in conventional CW EPR experiments.

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo.

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan electron-paramagnetic-resonance (RS-EPR), which can provide quantitative information under in vivo conditions on oxygen concentration, pH, redox status and concentration of signaling molecules (i.e., OH•, NO•). The RS-EPR technique has a higher sensitivity, improved spatial resolution (1 mm), and shorter acquisition time in comparison to the standard continuous wave (CW) technique. A variety of phantom configurations have been tested, with spatial resolution varying from 1 to 6 mm, and spectral width of the reporter molecules ranging from 16 µT (160 mG) to 5 mT (50 G). A cross-loop bimodal resonator decouples excitation and detection, reducing the noise, while the rapid scan effect allows more power to be input to the spin system before saturation, increasing the EPR signal. This leads to a substantially higher signal-to-noise ratio than in conventional CW EPR experiments.