250 GHz CW gyrotron oscillator for dynamic nuclear polarization in biological solid state NMR

Abstract

In this paper, we describe a 250 GHz gyrotron oscillator, a critical component of an integrated system for magic angle spinning (MAS) dynamic nuclear polarization (DNP) experiments at 9 T, corresponding to 380 MHz ¹H frequency. The 250 GHz gyrotron is the first gyro-device designed with the goal of seamless integration with an NMR spectrometer for routine DNP enhanced NMR spectroscopy and has operated under computer control for periods of up to 21 days with a 100% duty cycle. Following a brief historical review of the field, we present studies of the membrane protein bacteriorhodopsin (bR) using DNP enhanced multidimensional NMR. These results include assignment of active site resonances in [U-¹³C, ¹⁵N]-bR and demonstrate the utility of DNP for studies of membrane proteins. Next, we review the theory of gyro-devices from quantum mechanical and classical viewpoints and discuss the unique considerations that apply to gyrotron oscillators designed for DNP experiments. We then characterize the operation of the 250 GHz gyrotron in detail, including its long-term stability and controllability. We have measured the spectral purity of the gyrotron emission using both homodyne and heterodyne techniques. Radiation intensity patterns from the corrugated waveguide that delivers power to the NMR probe were measured using two new techniques to confirm pure mode content: a thermometric approach based on the temperature-dependent color of liquid crystalline media applied to a substrate and imaging with a pyroelectric camera. We next present a detailed study of the mode excitation characteristics of the gyrotron. Exploration of the operating characteristics of several fundamental modes reveals broadband continuous frequency tuning of up to 1.8 GHz as a function of the magnetic field alone, a feature that may be exploited in future tunable gyrotron designs. Oscillation of the 250 GHz gyrotron at the second harmonic of cyclotron resonance begins at extremely low beam currents (as low 12 mA) at frequencies between 320 and 365 GHz, suggesting an efficient route for the generation of even higher frequency radiation. The low starting currents were attributed to an elevated cavity Q, which is confirmed by cavity thermal load measurements. We conclude with an appendix containing a detailed description of the control system that safely automates all aspects of the gyrotron operation.

Introduction

Due to the excellent resolution of nuclear magnetic resonance (NMR) spectra, NMR has evolved as the preferred spectroscopic approach for the solution of problems in many areas of science, including physics, chemistry, biology, materials science, and more recently medicine. The excellent resolution is a consequence of long nuclear relaxation times that are in turn due to the small magnetic moments of the nuclear spins that couple weakly to the surrounding lattice. However, a deleterious effect of the small size of these magnetic moments is that the sensitivity of NMR experiments is low when compared to other spectroscopic approaches. Furthermore, since both high resolution solid state and solution NMR are utilized with increasing frequency in structural studies of macromolecular biological systems—proteins, nucleic acids, etc.—sensitivity continues to be an issue of paramount importance in the successful application of the technique.

Approaches to improving the sensitivity of NMR experiments have followed two avenues: innovations in instrumentation and developments in spectroscopic methodology. Outstanding examples of the former date from as early as the 1960s, when the appearance of laboratory computers enabled the implementation of Fourier transform NMR techniques resulting in signal-to-noise increases of 10–100 [1]. More recently, the development of superconducting magnets that operate at increasingly higher fields has significantly improved sensitivity, since the signal-to-noise per unit time scales as $\sim {\omega }_0^3$. Finally, in the last few years, cryogenically cooled probes with higher Qs in the r.f. coil and lower noise figures in the detection r.f. preamplifiers have become routinely available, improving the sensitivity by a factor of 1.5–3 depending on the conductivity of the sample [2].

Examples of innovations in spectroscopic methodology that have improved sensitivity are also numerous. Some of the most successful approaches involve polarization transfer techniques, including cross polarization (CP) in solids [3], [4] and INEPT transfers [5] in solution, in which the polarization of a spin with a large magnetic moment is transferred to one with a smaller moment. Today, CP is an integral part of high resolution magic angle spinning (MAS) experiments in solids [6] and multiple INEPT transfers are present in essentially every biological solution NMR experiment [7]. In these approaches, the sensitivity is enhanced by a factor of (γ_I/γ_S) or about 4 for I = ¹H and S = ¹³C and 10 when S = ¹⁵N. Another, and in fact the original, example of a polarization transfer experiment was proposed by Overhauser [8] and involved transfer of conduction electron polarization to nuclear spins in metals. Carver and Slichter [9], [10] verified Overhauser’s hypothesis that such transfers and signal enhancements were possible with low field (3.03 mT) experiments performed on samples of Li metal and other materials with mobile electrons. During the 1970s, the analogous nuclear Overhauser effect (NOE) was used extensively to increase sensitivity in spectra of low-γ species, and it is currently employed to estimate internuclear distances for structure determination by solution state NMR.

Extension of electron-nuclear and other high polarization transfer experiments involving noble gases, para-hydrogen, semiconductors, or photosynthetic reaction centers [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23] to contemporary solid state and solution experiments is very appealing, since it could significantly enhance the sensitivity in a variety of NMR experiments. In particular, the theoretical enhancement for electron-nuclear polarization transfers is approximately ∼(γ_e/γ_I), where now the ratio is ∼660, because of the large magnetic moment of the electron relative to the ¹H, making the gains in sensitivity large. Accordingly, during the 1960s and 1970s, there were extensive efforts to perform electron-nuclear polarization transfer in liquids [24], [25] and solids [26], [27]. All of these experiments, collectively known as dynamic nuclear polarization (DNP), require that the electron paramagnetic resonance (EPR) spectrum be irradiated with microwaves that drive the exchange of polarization between the electrons and the nuclear spins. In the case of liquids, these are Overhauser effect transitions and, in solids, other mechanisms—the solid effect (SE), thermal mixing (TM) or the cross effect (CE)—dominate the polarization transfer process. Since DNP experiments require irradiation of the EPR spectrum, they were confined to relatively low magnetic fields because of the paucity of high frequency microwave sources. In particular, the microwave sources used in both the liquid and solid state experiments were klystrons that operate at ⩽40 GHz, constraining DNP-MAS experiments to ⩽60 MHz ¹H frequencies. Thus, for DNP to be applicable to the higher fields employed in contemporary NMR experiments, new instrumental approaches to producing microwaves were required.

To satisfy these requirements, we turned to gyrotrons, a type of cyclotron resonance maser, as microwave sources for DNP experiments [28], [29], [30]. This choice was motivated by the fact that gyrotrons are fast wave devices [31], with interaction structures whose dimensions are large compared to the operating wavelength, and as such are capable of generating high powers (10–100 W CW) for the extended periods typical of multidimensional NMR experiments. Because the gyrotron interaction involves a resonance between the r.f. modes of an electromagnetic cavity and an axial magnetic field, the gyrotron frequency is in principle limited only by the available magnetic field strength. Furthermore, the cavity can be much larger than the operating wavelength, so the power density does not increase with the gyrofrequency, resulting in long lifetimes and high reliability. We anticipate that gyrotrons will be useful to at least the 1 GHz ¹H NMR frequency regime (∼660 GHz for electrons) or higher. For example, a gyrotron was recently operated in pulsed mode at a frequency of 1.03 THz [32].

In order to demonstrate the feasibility of employing gyrotrons in DNP experiments, we initially constructed a 140 GHz gyrotron oscillator that operates with a 211 MHz (¹H) NMR spectrometer [30], [33]. This system permitted us to demonstrate DNP at 5 T fields and to explore many important features of the experiments. For example, we established that cross effect DNP using biradical polarizing agents is the optimal mechanism [34], [35], [36], [37], [38], [39], [40], [41] for high field experiments involving CW microwave radiation. Traditional approaches, based on the solid effect and thermal mixing, yield enhancements that are an order of magnitude smaller [38] or require high concentrations of polarizing agents that lead to significant electron-nuclear dipolar broadening [42]. In addition, the 140 GHz system permitted us to develop and refine a number of experimental techniques, for example MAS at 90 K and lower temperatures [42], [43]. Finally, over the last few years, research with the 140 GHz system led to increases in DNP enhancements in MAS experiments from ∼10 to ∼300 [28], [35]. However, it remains that this spectrometer is operating at a 5 T field, and as such is a low frequency instrument by present day NMR standards.

In order to move DNP experiments to higher fields, we have built two additional gyrotron oscillators operating at 250 and 460 GHz, corresponding to 380 and 700 MHz for ¹H NMR, respectively. A cursory description of the 250 GHz oscillator appeared elsewhere [44] and the design and initial operation of the 460 GHz oscillator is described in detail in other publications [45], [46], [47], [48]. However, since the appearance of the brief description of the 250 GHz gyrotron, we have implemented many important changes to the system. In this paper, we characterize the operation of the 250 GHz gyrotron oscillator with detailed measurements of several important operating parameters. We have introduced two novel methods for imaging the millimeter wave beam and quantitatively determining its spectral purity. We have also demonstrated for the first time that the operating parameters of a gyrotron can be regulated under feedback control for indefinite and stable operation. In particular, this is the first gyrotron for DNP that operates continuously (in true CW mode), and we have achieved uninterrupted and regulated CW operation for a period of 21 days, a record for any gyrotron operating in this frequency regime. In addition, we have integrated the device into a low temperature solid state NMR spectrometer so that it now routinely performs multidimensional solid state NMR experiments on biological systems. Thus, the two primary goals of this paper are (1) to provide a detailed description of this new instrumentation for enhancing sensitivity in solid state NMR experiments and (2) to provide illustrative examples of the scientific possibilities afforded by the enhanced sensitivity available with this equipment. For the latter we present in this paper DNP enhanced MAS spectra of bacteriorhodopsin (bR), a 26.6 kDa membrane protein embedded in a lipid bilayer. This is a more challenging test case for the DNP method than small model compounds such as urea or proline that we have used extensively in other papers demonstrating DNP [36], [49], [50]. It is also a biologically important system that poses outstanding scientific questions and therefore addresses the applicability of DNP experiments to interesting systems.

An additional important aim is to familiarize members of the magnetic resonance community with high field DNP instrumentation. In particular, while gyrotrons are well known in the microwave community, they are virtually unknown in the magnetic resonance community. Thus, the contents of this paper will serve to familiarize the NMR and EPR communities with the rudiments of gyrotron technology and facilitate propagation of the instrumentation to other laboratories.

In Section 2 of the paper, we present DNP enhanced MAS NMR spectra of the membrane protein bacteriorhodopsin (bR) to demonstrate what is currently achievable with high frequency DNP experiments. These spectra include the first multidimensional spectra of a biological system acquired with DNP, and illustrate that it is possible to acquire spectra that are simply not accessible in the absence of DNP. Section 3 contains a brief discussion of the r.f. and microwave components of the 250 GHz/380 MHz DNP–NMR spectrometer. This includes the millimeter wave transmission line, low temperature MAS probe, and required cryogenics, although we defer complete descriptions of these three components to other manuscripts. Section 4 provides a detailed description of eleven major components of the 250 GHz gyrotron and their function including a discussion of the theory of the operation of gyrotron oscillators. Section 5 contains data on the operation of the gyrotron, including power output as a function of beam current, spectral purity and quality, frequency tuning as a function of the magnetic field, and frequency and power output stability as a function of several parameters. We also characterize the mode content in the millimeter wave transmission line through measurements of the radiated intensity pattern by liquid crystal thermometry and pyroelectric camera technology. Finally, in Section 6 we consider the possibility of second harmonic operation in which the gyrotron frequency is double that of the fundamental frequency in the same magnetic field. This is a feature of the 460 GHz system mentioned above since operation at the second harmonic generation will reduce the cost of the magnet associated with future high frequency gyrotrons.