In this tutorial lecture, I will discuss the fundamental principles, state of the art, and future opportunities, in advancing the sensitivity in ESR measurements. I will review (at a basic, non-technical level!) how the signal strength obtained in pulsed ESR can be understood from the point of view of ideas in quantum mechanics such as spontaneous emission, the Purcell effect, and Dicke super-radiance, and how this can help understand improvements in ESR sensitivity. I will examine how some basic ideas from ESR experiments employed in the context of quantum information and quantum technologies (including experiments performed at milliKelvin temperatures) can be applied to more typical ESR set-ups, offering substantial improvements in signal-to-noise ratio (SNR). 

By modifying a standard X-band ESR probe to include a cryogenic low-noise amplifier, we demonstrated SNR enhancements of 8x-15x (leading to a reduction in ESR measurement time of 60x-200x) for measurements at 4K [1]. These enhancements can be applied generally, and are compatible with typical experiments such as DEER, HYSCORE and ENDOR, as well as REFINE [2]. The same techniques can be applied at Q-band [3]. Quantum-limited cryogenic amplifiers offer the potential for even greater gains.

For samples which are limited in total spin number or geometry (e.g. spins localised on surfaces), a reduction in the resonator mode volume can yield many orders of magnitude increases in the spin number sensitivity [4]. The use of superconducting resonators is particularly beneficial in enabling high-Q cavities with mode volumes approaching femtolitres [4].

[1] M Šimėnas et al., J Mag Res 322 106876 (2021)

[2] K Richardson et al., Nature Communications 12 5387 (2021)

[3] V Kalendra et al., J Mag Res 356, 107573 (2023)

[4] A Bienfait et al., Nature Nanotechnology 11 253 (2015)

[5] V Ranjan et al., App Phys Lett 116 184002 (2020)

In the last decade, ESR moved to high fields and frequencies (HF-ESR) to achieve better resolution and sensitivity, and to get access to high-spin systems with large zero field splitting (also called single ion anisotropy). Nowadays, frequencies above 100 GHz have become standard. This work focuses on establishing a versatile broadband HF-ESR spectrometer using UCSB’s free electron laser (FEL) as a microwave source. Using either the ITST’s FEL (~kW power) or a low (~mW) power source from 170 GHz to 500 GHz and magnetic fields up to 16 T, our spectrometer will operate in pulsed, continuous wave, and rapid-scan modes. To enable pulsed FEL-ESR measurements we have developed and tested a new generation of modular quasi-optical pulse slicers for kW-power amplitude modulation. An additional component of the new FEL-ESR spectrometer design is a quasioptical sample holder that is able to significantly improve induction mode isolation and signal-to-noise (SNR) of continuous wave and rapid-scan ESR. Frequency-agile FEL-ESR will provide the opportunity to perform investigations of short-lived excitations at sub-THz frequencies that are otherwise inaccessible.

Johan van Tol, National High Magnetic Field Laboratory, USA

High-Frequency Electron (Para-)Magnetic Resonance has seen a relatively strong development since the early experiments in the seventies in the Lebedev group. High-Frequency EPR spectrometers are present in a number of university labs, but also in a few different user facilities where it serves researchers from a wide range of disciplines. We’ll go through some of the history and provide examples where high-frequency EPR has proven to be very useful. We’ll then focus on what the current cw- and pulsed EPR capabilities are for frequencies up to a THz and even beyond. We’ll also briefly touch on alternative detection/measurement techniques. 

Andreas Meyer, Max Planck Institute for Multidisciplinary Sciences, DE

Electron nuclear double resonance (ENDOR) probes transitions of nuclear spins coupled to nearby unpaired electron spins. Since almost all nuclei of the periodic table possess magnetic isotopes, many different ENDOR strategies can be devised to study paramagnetic biological molecules. This presentation is focused on 19F and 17O nuclei. 19F nuclei can be regarded as nuclear analogue of nitroxide spin labels to perform distance measurements in a distance range of ca. 0.7 – 1.5 nm. In recent months, we have worked to implement this approach at the widespread Q-band EPR set-up (1.2 T), which offers advantages in terms of sensitivity and resolution compared to the 3.4 T set-up used in most 19F ENDOR studies. The ubiquity of oxygen in biological samples renders 17O a valuable target for ENDOR studies. However, any measurement with 17O is complicated by the occurrence of quadrupole interactions, a low gyromagnetic ratio and low natural abundances. Despite this, we could detect ENDOR signatures of 17O nuclei at ca. 0.5 nm distance to the paramagnetic center, i.e. in its 2nd coordination sphere.

Austin McRae, North Dakota State University, USA

Enzyme encapsulation in Metal-Organic Frameworks (MOFs) can be challenging for large enzymes. Co-crystallization of MOFs in aqueous solution is a unique way to overcome this challenge, but the crystallinity of co-crystallized MOFs can be reduced. This drawback of co-crystallization raises concerns on how enzyme performance is impacted by the packing quality of these MOFs. In this work, multiple aqueous-phase co-crystallized MOFs are being further investigated utilizing site-directed spin labeling–EPR spectroscopy to probe enzyme dynamics and restriction. This technique probes protein backbone motions, which are directly related to the local crystal packing quality and density. This work suggests a connection between MOF crystal packing/density and protein mobility that could guide future design of materials for enzyme immobilization.

Song-I Han, Northwestern University, USA

Surface and internal water dynamics of molecules and soft matter are of great relevance to their structure and function, yet the experimental determination under ambient and steady-state conditions is challenging. We recently introduced a method to obtain local water dynamics through Overhauser dynamic nuclear polarization (DNP). The outcome of our approach is the quantitative determination of the key DNP parameter known as the coupling factor, which provides local translational diffusion dynamics of the solvent within 5 Å of the spin label. We discuss the discrepancy between the related field cycling relaxometry technique and DNP in determining the coupling factor and present arguments in support of the DNP-determined value. DNP measurements of local hydration dynamics around nitroxides in bulk water and on the surface of proteins are presented.

Lukas Woodcock, University of Denver, USA

A rapid scan EPR spectrometer operating at a microwave frequency of about 1 GHz achieves a spin sensitivity of ~2x1014 spins for an aqueous nitroxide sample in a 9 mm diameter resonator. The magnet and rapid scan driver and coils are similar to those reported by Buchanan et al. [1], but several changes were made to the system to minimize noise and loss in the detection system. These changes increase detection sensitivity. Changes included selection of sources and simplification of the signal detection path.

Jakub Hruby, National High Magnetic Field Laboratory, USA

Bottom-up chemical synthesis of molecular spin qubit architectures represents a novel way for pursuing next-generation quantum technologies that could substantially influence all fields of human activity from complex structural biology to finance. [1,2] Our work focuses on fine-tuning resonant clock transitions (CTs) within 4f n 5d 1 Ln(II) complexes, such that the associated transition frequencies, f, are insensitive to the local magnetic induction, B0 , with df/dB 0  → 0 at the CT minimum. This offers protection from magnetic noise and up to 10 times longer phase memory times, Tm, compared to conventional EPR transitions. [3] As an added bonus, hyperfine CTs associated with significant s-d mixing in 4fn5d1 Ln(II) complexes minimizes spin-orbit coupling, leading also to enhanced spin-lattice relaxation times, T 1 . [4]

Yifan Quan, Massachusetts Institute of Technology, USA

Recently, a theoretical and experimental study was presented that explains many aspects of the spin dynamics associated with the three microwave chirped pulse dynamic nuclear polarization (DNP) experiments. With an improved understanding of the spin dynamics of chirped pulsed DNP, we performed experiments using the 94 GHz HiPER spectrometer. Using chirped pulses, the polarization transfer efficiency can be optimized and an enhancement ε∼ 496 was observed using 10mM trityl-OX063 as the polarizing agent in a standard d 8-glycerol:D2O:H2O:6:3:1 glassing matrix at 70K. The frequency swept pulses enhance the nuclear magnetic resonance (NMR) signal, and also reduce the recycle delay, accelerating the NMR signal acquisition. Coherent pulsed DNP is still mostly limited at X-band and Q-band. We believe that our experimental results at W-band are a strong evidence that coherent pulsed DNP methods should be further developed at higher magnetic fields, where the NMR resolution can be yielded and chirped DNP is one of the most promising techniques at high fields.

John Franck, Syracuse University, USA

Water does not behave as a simple fluid, but rather as a complicated and coordinated mesh of dynamic hydrogen bonds. In particular, the water molecules that surround macromolecules store significant amounts of free energy, and water molecules at different sites display an unexpected range of properties. In this talk, we discuss how Overhauser Dynamic Nuclear Polarization can push magnetic resonance to its limits in order to interrogate the surprisingly complex water structures in what were previously thought of as the “white spaces” surrounding macromolecules.

Manoj Subramanya, National High Magnetic Field Lab. / Florida State Univ., USA

We report wideband Fourier transform (FT) EPR detection capabilities for a quasi-optical W-Band (94 GHz) spectrometer with 1 kW peak power and equipped with an arbitrary waveform generator. The spectrometer employs a non-resonant sample-holder with an instantaneous bandwidth of 1 GHz. Benchmark experiments are presented for the dilute standard TEMPOL radical, which consists of a 500 MHz wide EPR spectrum at W-Band. We demonstrate an efficient inversion of this broad inhomogeneous spectrum using a single adiabatic chirp pulse. Additionally, the FT-detection scheme was implemented for wide-band inversion recovery experiment and multi-dimensional EPR, demonstrating full capabilities of the spectrometer.

Francesco Torricella, National Institute of Health, USA

Studying biomolecules in their native environment represents the ideal sample condition for structural biology investigations. Here we present a novel protocol that allows delivering proteins into both prokaryotic and eukaryotic cells through a mild thermal stimulation. This method allows internalizing substantial amounts of proteins, with different molecular weight and conformation, without compromising the structural properties and cell viability. The data presented show the efficacy of this approach for delivering proteins in a concentration range suitable for successfully applying biophysical methods, such as cw-EPR and DEER spectroscopies.

Venkata SubbaRao Redrouthu, University of Konstanz, DE

Pulsed dynamic nuclear polarization (DNP) is a promising new approach to enhancing the sensitivity of high-resolution magic-angle spinning (MAS) NMR. The TOP (Time-Optimized Pulsed) DNP pulse sequence managed to deal with the matching condition problem at high fields by incorporating a modulation frequency. Based on a similar principle, we recently introduced the XiX (X-inverse-X) DNP sequence, which produced higher enhancement factors than TOP DNP in experiments at Q band (51 MHz/1.2 T/34 GHz) using moderate nutation frequency. We also explored the TPPM (Two-Pulse Phase-Modulation) DNP sequence, which is a generalization of XiX and has a larger parameter space. Our experiments clearly show that a DNP sequence with a larger scaling of dipolar coupling leads to a faster build-up of bulk nuclear polarization. In my talk, I will explain how this scaling comes about and how to determine it from trajectory analysis.

Katrin Ackermann, University of St. Andrews, UK

We show that PDS measurements down to 100 nM protein concentration for nitroxide-nitroxide PELDOR/DEER and 500 nM for CuII-CuII RIDME are possible with this experimental setup and standard labelling protocols, thus suggesting that sufficient concentration sensitivity can be afforded for applications in the sub-M regime. We hypothesised that it should be possible to reduce concentrations further with orthogonal CuII-nitroxide labelling. For this case, we could demonstrate that PDS provides dissociation constants for binding interactions at protein concentrations down to 50 nM in favourable cases. Exchanging the nitroxide with the trityl-based SLIM label afforded another boost in concentration sensitivity compared to the CuII-nitroxide case and yielded interpretable data down to protein concentrations of 10 nM. Notably, SLIM is stable in reducing environments, making this label combination a potential candidate for cell-based PDS studies.

Euan Bassey, UC Santa Barbara, USA

In this work, we present a combined high-frequency EPR and density functional theory (DFT) study of the local paramagnetic environments in Li 2 MnO 3 , a model compound for the family of commercially successful, layered lithium-ion battery cathodes. By collecting EPR spectra at a range of frequencies (9 – 383 GHz) and temperatures (5 – 300 K) and comparing to DFT-calculated g-tensors, we clearly observe several unique local environments and assign these observed resonances to Mn 4+ centres with different local environments [Figure 1]. We also examine the effect of magnetic exchange interactions on the spectra to explain the temperature evolution of these complex EPR spectra. The methodology presented in this work will be invaluable for studying paramagnetic centres in paramagnetic solids not only for LIBs and NIBs, but also in a range of TM-based materials for devices.

Nina Kubatova, National Institute of Health, USA

Despite extensive investigations on structure-function relationships of membrane G protein-coupled receptors (GPCR), the effects of membrane components, such as cholesterol and its derivatives, on oligomerization preferences remains to be clarified. Here, using DEER to measure distances between nitroxide spin labels, we show that the b1-adrenergic  receptor (b1AR) monomer-dimer equilibrium in dodecyl-b-D-maltoside micelles is modulated by sterols. Global fitting of DEER echo curves for spin-labeled b1AR upon titration with sodium cholate and cholesteryl hemissucinate demonstrates that saturation of micelles with the former induces receptor dimerization, while specific binding of the latter to b1AR inhibits dimerization and stabilizes the monomeric form. These results illustrate how quantitative analysis of DEER data can contribute to studies of GPCR oligomerization.

Guoquan Liu, Peking University, CN

Free radicals/ROS (reactive oxygen species) are not only profoundly implicated in the pathology of cancers, but also the active intermediates in several antitumor therapies. In this talk, we will first focus on the action mechanism of a conventional drug artesininin. Using electron paramagnetic resonance (EPR), we provided the first EPR spectrum of a speculated primary carbon radical generated after the activation of artemisinin, and identified a pH-dependent rearrangement of the artesininin derivatives that determines their antitumor activity. Secondly, we are inspired to develop a targeting strategy to drive photo-induced ROS to degrade selectively the protein of interest. With this photodegradation targeting chimera strategy (PDTAC), we have successfully degraded GPX4, a key protein regulating ferroptosis, and triggered potent antitumor immunity. In sum, our studies highlight the interesting role of radicals/ROS in developing new antitumor therapies.

Cooper Selco, University of Southern California, USA

We study optically addressable spin qubits in diamond, we can only experimentally probe the qubit and cannot directly observe interactions between the qubit and the environment. To model system-bath interactions, physicists most often use Markovian models where the spin-bath dynamics only depend on the current state of the system. However, it is possible for non-Markovian effects to be present in open quantum systems. Such models are said to include “memory effects” coming from the bath since the bath retains a memory of previous system states. We are working on techniques to experimentally probe the non-Markovian spin-bath dynamics present in single nitrogen-vacancy (NV) centers in diamond. Non-Markovian nvironmental bath dynamics can present a new perspective for understanding and combating quantum decoherence. Furthermore, this project gives new insights into a better understanding of the spin dynamics of open quantum systems.