DNP probe for high resolution, liquid nmr

Abstract

An NMR probe (1) for performing high resolution, liquid DNP-NMR, comprising: a corrugated waveguide (3) for mw transmission arranged along a longitudinal axis Z of the probe (1); a system of at least two mw mirrors (4-7) for mw beam transmission, focusing and shaping of the beam to match the sample geometry; at least one RF coil (11, 12) for NMR detection, mounted to allow the passage of mw to the sample region, characterised in that the cooling system comprises a flow Dewar tube (8) mounted at the side of the probe and parallel to the corrugated waveguide for feeding a cryogenic fluid, and an insulated sample chamber (26) surrounded by a chamber Dewar (9); a sample rotation device configured to achieve uniform irradiation on the sample volume to increase the sample volume under mw irradiation and reduce sample heating; a sample assembly (13) comprising concentric circular tubes or rods (15, 16) forming a constraint on the sample layer (14). This arrangement can excite large sample volumes, in particular even up to-40 [mu] L.

Description

Technical Field

[0001] The present invention relates to an NMR (=Nuclear Magnetic Resonance) probe head which is configured for performing high-resolution, liquid-state DNP (=Dynamic Nuclear Polarization)-NMR and comprises:

[0002] - a corrugated waveguide for mw (=microwave) transmission arranged along the longitudinal axis Z of the probe,

[0003] - a system of at least two MW mirrors for MW beam delivery, focusing and shaping to match the sample geometry,

[0004] - at least one RF coil for NMR detection, mounted in a way that allows the passage of mw to the sample area.

[0005] Such a DNP-NMR probe including associated MW and RF devices is described, for example, in Yoon, D., et al., High-Field Liquid-State Dynamic Nuclear Polarization in Microliter Samples. Analytical Chemistry, 2018. 90(9): p. 5620-5626 (ref.

[11] ).

[0006] Background of the invention and prior art

[0007] NMR is an established technique for obtaining structural information in chemistry, physics and molecular biology. The method is based on the detection of magnetic nuclei whose energy is split in a static magnetic field (denoted as B0). If such nuclei are exposed to radio frequencies (RF), resonant absorption can be observed and an NMR signal at the nuclear Larmor frequency can be recorded. However, the small energy splitting, and therefore the inherently small differences in the population of spin states (the so-called spin polarization), leads to limited NMR sensitivity. The lack of sensitivity can be improved by applying a stronger external magnetic field, however this is achieved to a limited extent and at very high cost. Another way to improve the sensitivity is the method known as dynamic nuclear polarization (DNP), see reference [1]. It exploits the large spin polarization of unpaired electrons, which can be transferred to nuclear spins using a double or multi-resonance setup, providing combined microwave (MW) and RF radiation. DNP provides impressive NMR signal enhancement in solids and is therefore widely used in solid-state magic angle spinning (MAS) and so-called solution DNP-NMR.

[0008] In liquids, DNP is dominated by the Overhauser effect (see references [2, 3]) and is performed by mixing a so-called polarizing agent (PA) (usually a nitroxide radical) with the target molecule of NMR. The electron spin resonance (ESR) transition of the PA saturates at the resonant microwave frequency, and the enhanced NMR signal of the target is detected in the RF range. Although NMR in liquids is the most widely used technique in NMR spectroscopy, DNP in liquids has not yet been established in NMR. The main reason is that one of the key parameters of the Overhauser effect is the saturation factor, which depends on several different parameters, but mainly on the microwave magnetic field intensity (B1) and therefore on the applied mW power. However, in liquids, especially polar solvents, microwave radiation is strongly absorbed. This effect is due to the high and frequency-dependent dielectric losses of most liquid media. This leads to strong sample heating (measured by the supplied mW power) on the one hand and a reduction in the B1 intensity on the other. Sample heating is particularly detrimental because it can destroy the molecules being studied and deteriorate the quality of the NMR spectrum. This problem can be alleviated to some extent by using microwave resonators, but these resonators require conductive materials in the immediate vicinity of the sample, which disrupts the electrostatic field uniformity. This results in a significant broadening of the NMR lines and, therefore, a loss of spectral resolution. In addition, microwave resonators significantly reduce the sample volume, offsetting the sensitivity gain. The latter disadvantage is particularly severe at high magnetic fields (such as those used in the present invention) because the size of the resonator (measured by the wavelength of the microwaves used) is significantly reduced.

[0009] Another challenge arises from the intrinsic DNP mechanism, which is based on cross-relaxation between electron and nuclear spins, which decreases in efficiency as the static magnetic field B0 increases. Moreover, cross-relaxation is often driven by two mechanisms (dipolar and scalar relaxation), delivering enhancements of opposite signs and thus canceling each other, especially at low and medium magnetic fields, where dipolar interactions are still quite high. However, we and others have recently demonstrated that the scalar mechanism operates at high magnetic fields, which is relevant for modern NMR, see references [3–8]. Therefore, the establishment of liquid DNP is currently limited by the availability of suitable probe designs.

[0010] Attempts to provide high-field / frequency, liquid-state DNP-NMR probes have been described in references [9–15]. These reports disclose valuable designs and technical solutions, but are primarily suitable for efficient microwave excitation, not for high-resolution NMR. Therefore, even though they report interesting DNP mechanistic results, these probes cannot be used for routine high-resolution NMR spectroscopy. In particular, resonator-based designs exhibit the aforementioned drawbacks of small sample volumes and loss of spectral resolution, as seen in references [12–14, 16, 17].

[0011] For example, reference

[11] already cited above describes a planar probe suitable for 31 Liquid DNP on P and protons at 9.2 T, however, is only suitable for relatively small sample volumes. Despite being a non-resonant configuration, it provides reasonably high B1 intensities at high input powers (≈2.5 G at 70 W input). The irradiated sample volume is close to 5 μL (considering the reported beam waist of 5 mm and a penetration depth of ≈60 μm in water at 5°C), while the total sample size detectable by NMR is about 10 μL. In addition, effective sample cooling is provided by the use of a gold-plated high thermal conductivity AlN support. Even though the probe shows high microwave and cooling efficiency, it contains an excess of conductive material in the immediate vicinity of the sample. As a result, the reported proton NMR line widths are in the range of ∼10 ppm at 9.2 T, which is far from the current high-resolution NMR standard.

[0012] A similar device is described in reference [9]. It comprises an RF transducer for NMR detection, which forms a quasi-resonant Fabry-Perot (FP) configuration together with a gated transistor that is partially transparent to microwaves. The device is instructive and can be fabricated in different arrangements, however, as in the previous example, the inclusion of a conductive material close to the sample leads to disturbances in the field homogeneity.

[0013] This is also true for reference

[10] , which describes another resonant device in which NMR detection is provided by a conductive stripline that simultaneously forms part of a Fabry-Perot resonator. Despite the dual-resonance configuration and the unconventional RF coil design, the probe is characterized by both high sensitivity for NMR and high microwave efficiency for DNP. Furthermore, this design is beneficial because it exhibits improved sample cooling capabilities. However, it again uses field-interfering materials close to the sample, and the sample volume is strongly reduced. Consequently, this device is limited to specialized applications.

[0014] To overcome these drawbacks, we have designed a liquid DNP-NMR probe that allows microwave excitation of large sample volumes, yet maintains optimal NMR performance, i.e., controlled sample heating, and without compromising NMR resolution. Our recent work (see references [4, 7]) and reports from other groups (see references [6, 16]) suggest that the B1 magnetic field required to saturate the EPR transitions of conventional polarizers should be in the range of 2–3 G. This magnetic field can also be achieved in a non-resonant microwave setup using a high-power source (e.g., a gyrotron, with an output power >10 W), but requires a special sample geometry to allow microwave penetration; i.e., the sample thickness must be less than the microwave penetration depth. Furthermore, efficient cooling must be implemented to balance sample heating. In this context, a non-resonant microwave setup is advantageous because it allows large sample volumes and does not require the introduction of field-interfering materials (i.e., metals), especially in the immediate vicinity of the sample. Such a setup should not compromise NMR resolution and should be easier to handle in routine experiments. The primary objective of the present invention is to provide a DNP probe for both routine and high-resolution NMR spectroscopy.

[0015] Purpose of the present invention

[0016] One object of the present invention is to provide a liquid DNP-NMR probe for conventional NMR that is capable of exciting large sample volumes, in particular up to 40 μL. Preferably, the probe should be designed to operate at microwave frequencies close to 263 GHz used for DNP and should be suitable for standard double-resonance NMR at fields close to 9.4 T. However, the design approach should be readily adaptable to operation at higher mW frequencies (up to THz) and NMR fields.

[0017] It would further be preferred to provide a DNP-NMR probe head in which the microwave components, in particular the focusing mirrors, and the sample assembly do not contribute to inhomogeneities in the static magnetic field, thus allowing high-resolution NMR measurements.

[0018] Furthermore, it is an object of the present invention to provide a DNP-NMR probe in which the mw is transmitted through a liquid sample and the temperature of which can be controlled to avoid boiling of the sample.

[0019] Finally, it is an object of the present invention to provide a DNP-NMR probe in which the sample can be rotated during the experiment, thus significantly increasing the effective sample volume exposed to microwaves. Summary of the Invention

[0020] These objects are achieved by designing a universal NMR probe as defined above on the first page, characterized in that

[0021] a cooling system comprising a flow Dewar tube for feeding a cryogenic fluid mounted at the side of the probe and parallel to the corrugated waveguide and an insulated sample chamber surrounded by a chamber Dewar,

[0022] - a sample rotation device configured to achieve uniform illumination over the sample volume to increase the sample volume under MW illumination and to reduce sample heating,

[0023] - A sample assembly comprising concentric circular tubes or rods forming a constraint for the sample layer.

[0024] The DNP-NMR probe head according to the invention is particularly optimized for high-resolution DNP-NMR, which operates at the EPR / DNP frequency, in particular close to 263 GHz.

[0025] To perform DNP, the probe includes a microwave component consisting of a corrugated inverted cone, a corrugated WG (waveguide), and a microwave mirror system. The MW (microwave) beam from the source is transferred via the cone and the corrugated WG to the mirror system for focusing, transforming, and further directing the MW beam to the sample assembly. The mirror is mounted within the thermally insulated sample chamber, close to the sample assembly and NMR coil.

[0026] The sample arrangement is considered to be a key subject of the present invention.The liquid sample is confined by two concentric tubes or rods of circular cross section and preferably made of quartz, forming a thin layer whose thickness is chosen depending on the dielectric properties of the sample and does not exceed the mw penetration depth.

[0027] Furthermore, the sample assembly is rotated about the Z-axis, primarily to increase the sample area exposed to the microwaves and secondly to prevent overheating of the sample during exposure. According to the present invention, the sample rotation apparatus is configured to achieve uniform illumination across the sample volume, thereby increasing the sample volume exposed to the microwaves and reducing sample heating. This sample rotation method increases the effective sample area exposed to microwaves and improves cooling efficiency under microwave excitation.

[0028] Furthermore, the use of a thin sample layer confined by a tube or rod leads to an enhancement of the B1 field at the sample location, which can be reasonably explained by the increased standing wave ratio caused by interference with the reflected wave at the interface of two dielectric media with different permittivity values.

[0029] To further protect the sample from overheating, the probe houses a cooling unit designed to deliver a cryogenic fluid, such as cold N₂ gas, to the sample. This unit comprises a dewar, preferably made of glass, and an insulated sample chamber. The cryogenic fluid is injected into the dewar, distributed within the sample chamber, and removed from there to provide a constant flow over the sample surface.

[0030] In essence, the present invention has the following advantages:

[0031] 1) The probe is suitable for high-resolution DNP-NMR on large liquid sample volumes which are still protected from overheating.

[0032] 2) The design allows microwave excitation without causing magnetic field inhomogeneities, so NMR resolution is not affected.

[0033] 3) The MW and cooling unit can be easily installed in existing standard liquid NMR probes, thus providing opportunities for a wide range of DNP-NMR measurements.

[0034] Preferred embodiments and further improvements of the present invention

[0035] The thickness of the relatively thin sample layer in the NMR probe according to the present invention can vary depending on the dielectric constant ε of the solvent, the sample temperature, and the mw frequency. In a preferred embodiment, the thickness of the sample layer is between 0.25Δ and 1.25Δ, where Δ is the penetration depth of the mw in the liquid sample medium.

[0036] Preferably, the thickness of the sample layer does not exceed the penetration depth Δ of mw in the liquid sample medium.

[0037] In a particularly preferred embodiment of the NMR probe according to the present invention, the sample assembly comprises two concentric circular tubes or rods. This arrangement is advantageous because it allows for the formation of a sample layer of desired thickness, not exceeding the MW penetration depth Δ, and depending on the dielectric properties of the sample. Furthermore, the cylindrical sample geometry is advantageous when rotating the sample about the Z-axis, which is particularly advantageous in order to: 1) increase the sample volume excited by the MW and 2) reduce sample heating.

[0038] In a preferred further development of this embodiment, two or more concentric tubes or rods may be used to have more than one sample layer, each sample layer having a thickness of no more than Δ / n (n being the number of layers), and thus further enlarging the sample volume.

[0039] In a preferred further development of this embodiment, the sample assembly is made of a material with low MW absorption, preferably quartz or any MW transparent material. This is required to 1) minimize MW losses and 2) further reduce sample heating due to losses in the assembly tube (rod).

[0040] Also advantageous are embodiments in which four MW mirrors are provided that transmit MW to the sample assembly while maintaining appropriate B1 magnetic field polarization and forming a beam profile that is optimally distributed across the sample geometry to reduce heating thereof and increase the efficiency of MW excitation. An arrangement having four MW mirrors allows the use of a design in which the corrugated waveguide assembly is mounted along the Z-axis, which is advantageous when considering space limitations in NMR probes.

[0041] In a preferred embodiment, the mirrors have a curvature based on a conical section, or they can be shaped as a quasi-ellipse, particularly with a curvature that provides efficient focusing and beam passing. In addition, the mirror curvature and its relative arrangement are designed in a way that reshapes the radial beam profile to match the sample geometry.

[0042] In another embodiment of the present invention, the MW mirror system for MW beam transmission, focusing and shaping the beam includes four mirrors located at distances of 0 mm, 17.25 mm, 42.5 mm and 34.50 mm from the Z axis (the last two values are measured at the height of the coil center) to form a beam profile that is optimally distributed over the sample geometry. A further advantage is that all four mirrors are mounted far enough away from the sample assembly and do not contribute to static field inhomogeneities at the sample location.

[0043] Another preferred embodiment provides Ceramic-made mirrors, which are specifically coated with gold, are used to minimize magnetic field distortion around the sample and maintain high homogeneity of the static magnetic field B0.

[0044] In a preferred further development of this embodiment, the coating of the mirror has a thickness between 3δ and 7δ, where δ is the skin depth in gold at room temperature (~0.15 μm), in particular to maintain a high conductivity of the skin layer for mW frequencies close to 263 GHz, but still minimize magnetic field distortion around the sample. The use of gold offers the further advantage that the thickness of the metal layer can be reduced due to the material's high conductivity and high oxidation resistance.

[0045] Also advantageous are embodiments in which the mirrors are designed to transmit the MW beam with an appropriate B1 magnetic field polarization orthogonal to the static magnetic field B0 to saturate the ESR transition of the DNP, and are designed to minimize magnetic field distortion around the sample and maintain high uniformity of the static magnetic field B0 required for high-resolution NMR.

[0046] In another embodiment of the present invention, two RF coils are provided, one of which is matched for a second resonant frequency to measure other nuclei or for 2H locking. Furthermore, the use of two RF coils is advantageous as it allows performing nuclear coherence 2D experiments and heteronuclear decoupling.

[0047] Further preferred are embodiments in which at least one shielding tube is arranged concentrically around the sample assembly to guide the cryogenic fluid. In particular, two shielding tubes are provided to support the NMR coil and are configured to increase the efficiency of sample cooling. The shielding tubes also prevent potential vibration of the coil under airflow.

[0048] In a particularly preferred embodiment of the present invention, the corrugated waveguide is designed to minimize MW losses in the desired frequency range and maintain the desired polarization of the microwave magnetic field B1. In addition, the waveguide is designed in a way that minimizes the distortion of the B0 magnetic field around the sample, i.e. it is installed far enough away from the sample area.

[0049] Particularly preferred is an embodiment of the invention, wherein the cooling system comprises means for controlling the temperature of the sample in order to minimize heating of the sample during mw excitation and to stabilize the temperature of the sample at a specific value.

[0050] Also within the scope of the present invention are embodiments designed for high-resolution 1D and 2D DNP-enhanced NMR spectroscopy of magnetic nuclei exhibiting the Overhauser effect using free radical polarizers, particularly using non-resonant microwave devices. Non-resonant devices will not affect NMR resolution and are easier to handle in routine experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Further details and advantages of the present invention will be described below with reference to the accompanying drawings, which show:

[0052] Figure 1 A perspective view of a DNP-NMR probe head according to a preferred embodiment.

[0053] Figure 2 Simplified diagram of the microwave and cooling components of a DNP-NMR probe.

[0054] Figure 3 Simplified view of the corrugated WG assembly and reflector arrangement.

[0055] Figure 4 Cross-sectional view of the mirror and sample arrangement indicating the microwave path and sample exposure.

[0056] Figure 5 Simplified cross-sectional view of the mirror arrangement showing the microwave path and sample exposure.

[0057] Figure 6 Simplified view of the cooling components including the thermally isolated sample chamber.

[0058] Figure 7Cross-sectional view of a thermally isolated sample chamber with indication of N2 gas flowing around the sample assembly.

[0059] Figure 8 CST model used for microwave simulation (a), and different cross-sectional views of the microwave B1 field pattern at 263.3 GHz and phase φ = 0 when simulating with water as the solvent (b), (c), and (d).

[0060] Figure 9 The NMR coils and their arrangement relative to the microwave beam are shown as a z-axis orthogonal section through a CST simulation of a water sample.

[0061] Figure 10 The NMR coils and their arrangement relative to the microwave beam are shown as a Y-axis orthogonal section through a CST simulation of a water sample.

[0062] Figure 11 Graph indicating the predicted microwave field intensity along the Z-axis at the front and back sides of the sample when simulated with three different solvents (CCl4, CHCl3 and water).

[0063] Figure 12 In with 15 Nd 16 -4-oxo-TEMPO was recorded in a mixture containing CCl4 and 13 Boltzmann and DNP enhanced spectroscopy on both CHCl3 samples 13 C-NMR spectrum. Sample composition: 200mM 13 CHCl3 was dissolved in 15 Nd 16 Natural abundance of 4-oxo-TEMPO in CCl4. NS represents the number of scans. Upper inset: applied pulse sequence.

[0064] Figure 13 a) Applied 1 H decoupling DNP pulse sequence; b) CCl4 doped with 25 mM 15 Nd 16 Boltzmann (mw off) and DNP-enhanced (mw on) spectra of ∼500 mM naturally abundant fluorobenzene of -4-oxo-TEMPO, with the magnified view in c) C ortho and d) C meta NS represents the number of scans.

[0065] Figure 14 Under DNP conditions, CCl4 13 Total correlation (TOCSY) 2D spectrum on C6-C6HC5I: a) represents C ipsoPosition of magnetization transfer 13 Structure of C6-C6H5I; b) TOCSY pulse sequence; c) 2D DNP TOCSY spectrum, where the 1D DNP spectrum of the same sample measured independently is used as a guide in the ω1 and ω2 dimensions; d) Boltzmann TOCSY spectrum, where the 1D Boltzmann spectrum of the same sample measured independently is used as a guide in the ω1 and ω2 dimensions. Sample composition: ~500mM 13 C6-C6H5I and ~25mM 15 N-TN-d 16 NS represents the number of scans.

[0066] Figure 15 Sodium pyruvate-3- 13 Boltzmann (mw off) and DNP-enhanced (mw on) C in water 13 C-NMR spectra: glycerol (9:1) and 25 mM 15 Nd 16 -4-oxo-TEMPO mixture. NS represents the number of scans. Upper inset: applied pulse sequence. DETAILED DESCRIPTION

[0067] The present invention relates to an NMR probe (1) comprising microwave reflecting and focusing mirrors (4, 5, 6, 7) for performing DNP-enhanced NMR in liquids. More specifically, the present invention relates to an NMR probe configured for conventional high-resolution DNP-NMR on large (tens of microliters) liquid sample volumes. Furthermore, the probe comprises an efficient cooling system (8, 9, 10) to mitigate sample heating during microwave excitation.

[0068] The DNP-NMR probe according to the present invention is particularly optimized for high-resolution DNP-NMR, which operates at an EPR / DNP frequency close to 263 GHz. The design is based on a commercially available wide-bore, dual-channel liquid NMR probe (Bruker Biospin). For NMR detection, the probe comprises two saddle coils (11, 12) close to the sample. The coils can be tuned and matched to the sample on both channels. 1 H. 19 F. 31 P. 13 C. 15 N and 2 The resonant (Larmor) frequencies of H are ν≈400 MHz, ≈390 MHz, ≈162 MHz, ≈100 MHz, ≈41 MHz and ≈61 MHz respectively at a given NMR field B0 = 9.4 T. The sample tube assembly (13) is placed axially with respect to the static magnetic field B0 in the NMR coil.

[0069] To perform DNP, the probe comprises microwave components, including a corrugated downtaper (2), a corrugated WG (= waveguide) (3) and a system of 4 (four) microwave mirrors (4, 5, 6, 7). The mw beam from the source (here a gyrotron) is transferred via the cone (2) and the corrugated WG (3) to a system of mirrors (4, 5, 6, 7) that is used to focus, convert and further deliver the microwave beam to the sample assembly (13). Specifically, the sample is placed in a narrow sample geometry (≈8×5 mm) 2 ) and expand the microwave energy to the input Gaussian beam (TEM 00 mode) into an elongated beam by designing a system of reflectors (4, 5, 6, 7) (see Figure 2 , 4, 5, 8). The mirrors (6) and (7) are mounted inside the thermally isolated sample chamber, close to the sample assembly (13) and the NMR coils (11, 12).

[0070] This sample arrangement is considered to be a key theme of the present invention. The liquid sample is confined by two concentric quartz tubes (15, 16) to form a thin layer, the thickness of which is selected according to the dielectric properties of the sample and does not exceed the mw penetration depth. In addition, the sample assembly is rotated around the Z axis, which is first implemented to increase the area of the sample exposed to the mw and secondly implemented to avoid overheating of the sample exposed to the mw. In addition, the use of a thin sample layer confined by the quartz tubes leads to an enhancement of the B1 field at the sample position, which can be reasonably explained by the increased standing wave ratio caused by interference with the reflected wave at the interface of two dielectric media with different dielectric constant values.

[0071] In order to additionally protect the sample from overheating, the probe houses a cooling unit (8, 9, 10; 26) designed to deliver cold N2 gas to the sample. It consists of a glass Dewar (8) and an insulated sample chamber (26). The cold N2 gas is injected into the Dewar (8) through the Dewar's inlet (25), distributed inside the sample chamber (26) and discharged through the chamber outlet (10), preferably realized as a discharge cup at the top of the chamber to provide a constant flow above the sample surface (see Figure 6 ).

[0072] The general design of the DNP probe (1) is shown in Figure 1 A cross section and illustration of the assembled probe show details of the assembly. Further details of the main components are given in Figure 2-7 Overview in.

[0073] The basic structure of the probe (1) can be described as a standard liquid NMR probe, which is combined with microwave components (2, 3, 4, 5, 6, 7) and cooling systems (8, 9, 10) to pump the EPR transition and stabilize the sample temperature, respectively (see Figure 1-6 ).

[0074] For NMR detection, 2 (two) saddle coils (11, 12) are mounted around the sample assembly (13). The axes of the coils are perpendicular to the main magnetic field (B0), which is oriented along the detector axis (axis Z). The larger coil (11) can be tuned to 1 H and 19 The resonant frequency of F (v = 380–400 MHz). The second smaller coil (12) is designed to be tuned at 13 C (~100MHz). It can also be tuned to 2 The frequency of H (~61 MHz) can therefore be used for frequency domain locking. Matching to other NMR frequencies is possible after minor modifications to the tuning circuit.

[0075] The central hollow column of the probe is formed by a corrugated waveguide (3) assembled from 11 shorter segments to form a total length of -435.5 mm (see Figure 1-3 Each WG segment is made of German silver and corrugated with corrugation parameters p = 0.30 mm, d = 0.28, w = 0.15 mm, a = 3.8 mm, where p, d, w, and a are the period, groove depth, groove width, and waveguide inner radius, respectively. On the input side, the WG (3) is joined to a corrugated cone (2), which couples the WG to the transmission line from the microwave source. The input inner diameter of the cone is 19.3 mm. The output diameter is equal to the inner diameter of the corrugated WG (3), which is 7.6 mm.

[0076] Microwave beam in corrugated WG as hybrid HE 11 mode propagates, so at the output it is efficiently coupled to the TEM 00 The free-wave mode, in the form of a beam with an approximately Gaussian profile, is further directed to the sample by a system of three mirrors with a curvature based on a conic section (4, 5, 6). The mirrors are arranged in such a way that the sample is exposed to the mw beam from one side (see Figure 4 ). Mirrors (4) and (5) are used to propagate the beam from the aperture of the WG and focus it to the beam waist between mirrors (5) and (6). The beam profile has approximately cylindrical symmetry throughout the beam path. Mirror (6) then refocuses the beam and reshapes the beam profile, focusing it to an elongated beam waist at the sample. This elongated profile covers the volume of the sample, which generates NMR signals more efficiently than a cylindrically symmetric beam. In addition, the mirrors are arranged in a way to maintain the polarization of the input microwaves, with the orientation of B1 of the input microwaves being perpendicular to the static magnetic field B0 and therefore perpendicular to the Z axis (see Figure 4Finally, a reflector (7) is located behind the sample volume to reflect residual microwaves. Additional precautions are taken to minimize the cross-polarization component in the reflected beam, which is mainly defined by the curvature of the reflectors. In a preferred embodiment, the degree of curvature of the reflectors can be described by their focal lengths, which are 24.4 mm, 33.3 mm, and 18.1 mm (maximum) for reflectors (4, 5, 6), respectively.

[0077] An important feature of the present invention is that the reflector bracket is made of The NMR spectrometer is made of ceramic, and only the reflective surface is coated with gold. The thickness of the metal layer is approximately 1 μm, which is several times greater than the skin layer in gold (~0.15 μm) at 263 GHz. This method is used to minimize magnetic field inhomogeneities at the sample location, which are typically introduced by metals and degrade NMR resolution.

[0078] The sample tube assembly (13) is positioned along the Z axis and inside the NMR coil ( Figure 2 and 4 ). Sample arrangement is considered to be another key subject of the present invention. The liquid sample (14) is confined by two quartz tubes (15, 16) so as to form a thin layer with a certain thickness, which is selected according to the dielectric properties of the sample (see Figure 4 ). It is essential that the layer thickness is less than the mw penetration depth in the sample medium. In a preferred embodiment, the dimensions of the outer QZ tube (15) are 4.97 mm (outer diameter (OD)) and 4.21 mm (inner diameter). The OD of the inner QZ tube (16) varies between 4.16 mm and 4.06 mm depending on the sample. The sample assembly (14, 15, 16; also referred to as 13) is rotated along the Z axis (see Figure 4 ), which is required for two key reasons: 1) to increase the area of the sample exposed to the microwaves, in particular to the maximum B1, and 2) to avoid overheating of the sample volume under microwave radiation. The first reason is particularly important because it represents a way to increase the sample volume under microwaves and its excitation uniformity, and is therefore also considered to be a key method of the present invention. Since DNP irradiation times reach several seconds, or in some experiments even continuous wave irradiation, a sample tube that spins slowly at a frequency of about 20 Hz is applied in this embodiment. The rotation of the sample is performed using the standard capabilities of a commercially available Bruker detector (Bruker Spinner). There are two other quartz tubes (17, 18) near the sample. They are implemented to stabilize the NMR coil and improve the cooling airflow around the sample.

[0079] The implementation of a thin sample layer (14) confined by two quartz tubes (15, 16) allows the layer thickness to be kept below the mw penetration depth. Furthermore, an important aspect of this configuration is the ability to increase the B1 intensity at the sample location due to interference at the interface of two dielectric media with different dielectric constants.

[0080] The microwave capabilities of this embodiment are achieved by using the CST Microwave Studio Suite TM )2019 (Dassault Systemes) finite element numerical simulation was used to verify. For the simulation, the application Figure 8 The simplified model shown in (a) consists of a corrugated WG segment, a reflector (4, 5, 6, 7), a PTFE window (21), an NMR coil (11, 12), a sample assembly (13) and a shielding tube (17, 18). The model is connected to the WG segment through a linearly polarized Gaussian beam (TEM) at the port (simulating the probe input). 00 mode) was fed. The input power of the Gaussian beam was set to 20 W. The MW polarization was similar to the beam polarization provided by the microwave source (here, a gyrotron). Simulations were performed to verify the design of appropriate beam alignment, polarization, and beam transformation properties. Different solvents were used to evaluate sample-specific microwave losses and B1 intensity at different sample locations.

[0081] Figure 8 (b, c) show the B1 intensity wave pattern at a specific phase moment (here, phase φ = 0°) in the front and back sections (YZ plane) of the sample, respectively. These figures show the elongated field distribution of the MW beam, as expected to expose the geometry of the NMR-active sample. Figure 8 (d) shows the same simulation, but in the XY-cut plane, at a point of height at the center of the coil along the Z axis, while Figure 9 Shown with Figure 8 (d) The same cut, but at a different viewing angle, better illustrating the coil arrangement relative to the beam profile. This arrangement is specifically designed to facilitate efficient passage of microwaves to the sample (14). Figure 10 This coil arrangement and the beam profile in the XZ-cut plane are shown.

[0082] Figure 11 The figure shows the front and back sections (YZ plane, see reference ) for three different solvents (CCl4, CHCl3 and water). Figure 8Figures (b, c) show the estimated B1 intensity over the active sample height (0-22 mm) along the Z axis. The cut section is orthogonal to the beam propagation and is formed at the midpoint of the sample layer, with thicknesses of 74 μm, 40 μm, and 25 μm for CCl₄, CHCl₃, and water, respectively. Simulations demonstrate that the probe's MW arrangement provides effectively strong B1 fields, approaching the values required to saturate the PA (≈2-3 G), but with reasonably high input power (≈20 W).

[0083] Another important feature of the present invention is the cooling of the sample assembly (13) under microwave irradiation. The cooling unit (8, 9, 10) is configured to deliver cold N2 gas to the sample position. It consists of a glass Dewar flask (8) mounted parallel to WG (axis Z) and an insulated sample chamber (cryostat) (26) surrounding the sample assembly (13) (see Figure 5 ). The sample chamber (26) consists of a chamber glass dewar (9) and an outlet (10). Cold N2 gas is injected into the inlet (25) of the transfer dewar (8) and then enters the cryostat through the entrance (20) to cool the sample. The heated gas is then removed through the outlet (10) to provide a continuous gas flow in the sample area. Using a commercial Bruker VTU N2 gas cooling unit, the gas temperature at the inlet (20) can be stabilized and controlled over a wide range, which for the present invention is 180-320K. Cooling can also be achieved using a Bruker BCU, which does not require the use of a liquid N2 evaporator. A PTFE window (21) is used to isolate the sample chamber and allow microwaves to pass from the reflector (5) to the reflector (6).

[0084] The given embodiments of the probe have been tested on different samples using DNP experiments performed at near room temperature. Experiments were performed using a Bruker AVANCE Neo 400 MHz NMR console and a custom-made 263 GHz gyrotron as a microwave source. Samples were prepared by deoxygenation in a glove box and by freeze-pump-thaw cycles (4, 5) with efficient O removal. The gyrotron frequency was set to resonate with the nitrogen oxide low field lines.

[0085] As an example, Figure 12 shows that when doped with 10 mM 15 Nd 16 200 mM of the naturally abundant 4-oxo-TEMPO in CCl4 13 The model system of CHCl3 was obtained 13 C NMR signal enhancement (ε), the effectively irradiated sample volume is ∼25 μL at a calibration sample temperature of 300 K. The actual sample volume required to be detectable by NMR and maintain high homogeneity is ∼40 μL. Figure 12 The upper inset in shows the pulse sequence used for DNP. We observe 13 C enhanced to 120 ( 13 CCl4) and ~200( 13 CHCl3), and remarkably, there is no loss of NMR resolution. When DNP irradiation is done in continuous wave mode, rotation of the sample assembly (13) is applied (frequency 20 Hz). In addition, the sample temperature is controlled by a flow of N2 provided by a cooling unit (8, 9, 10; 26). It has been demonstrated that the mw component of the probe does not disturb the B0 field homogeneity and that sample heating can be efficiently compensated by cooling. As a result, the NMR lines under DNP excitation show a significant enhancement and only limited broadening compared to the lines in a standard liquid NMR detector.

[0086] Another evaluation of NMR resolution is Figure 13 (b, c, d) are given, which show that the CCl4 is doped with 25mM 15 Nd 16 Enhancement (mw on) and Boltzmann (mw off) spectra of ~500 mM natural abundance fluorobenzene of -4-oxo-TEMPO. Figure 13 (a) shows the applied pulse sequence. During the detection, 1 H presaturation is used for decoupling. Experiments have shown that both DNP and Boltzmann spectroscopy exhibit high NMR resolution (LW≈2.3 Hz), enabling the detection of 2 JCF ≈ 21 Hz and 3 The DNP experiments were performed at a microwave power of 43 W and the sample temperature was kept in the range of 290–310 K.

[0087] An important feature of any NMR setup is the ability to perform multidimensional experiments, which provide insights into spin-dependent interactions between neighboring nuclei. Such experiments are typically performed over many hours, and the stability of the experimental conditions, including the sample temperature and its structural fidelity, is an important factor in determining the experimental results. To verify this factor under DNP conditions, the stability of the NMR spectrum was recorded in the presence of ∼25 mM NMR. 15 Nd 16 -4-oxo-TEMPO in CCl4, 13 C6-C6H5I( Figure 14 (a) 2D total correlation (TOCSY) spectrum on (a). Figure 14(b) Depicts the pulse sequence of the experiment. In DNP-TOCSY, polarization transfer between adjacent nuclei occurs during a period of isotropic mixing (performed synchronously with the mw excitation), where a spin-locked pulse train consisting of multiple π pulses is applied. The efficiency of this process is different for each cross peak and depends firstly on the DNP effect and secondly on the mixing time τm. In this experiment, DNP was performed under continuous wave (CW) irradiation with an mw power of ≈43 W and a τm of 20 ms. The sample temperature was maintained in the range of ≈290-310 K. In addition, the τm during both DNP and Boltzmann measurements was used. 1 H-decoupling. Figure 14 (c and d) show 13 DNP and Boltzmann TOCSY spectra of C6-C6H5I. Obviously, after DNP, all diagonals and cross peaks are observed. 13 The C signal was enhanced. Moreover, the observed enhancements were comparable to those obtained in the corresponding 1D experiments, demonstrating that the experimental conditions were stable enough to perform 2D DNP experiments.

[0088] at last, Figure 15 Shown in a mixture with 25mM 15 Nd 16 -4-oxo-TEMPO in water: sodium pyruvate in glycerol (9:1) 13 DNP on C. Due to the high dielectric losses in aqueous solvent, the sample layer and its volume were reduced by a factor of two to 25 μm (front and back layer thickness) and ~8 μL, respectively. The DNP-NMR spectra were recorded after 4 s of excitation with ~23 W mw power at a preset sample temperature of 275 K and delivered an enhancement of nearly 3. The spectra showed fairly good line widths, which were 9.6 Hz after DNP compared to 9.9 Hz in the Boltzmann spectrum. Since pyruvate is a well-known component used to track metabolism in cancer cells, it is important to provide DNP-NMR capabilities for this type of molecule, which, however, is only efficiently soluble in water. Our experiments show that DNP in water is also feasible using the new probe.

[0089] Summarize

[0090] A dynamic nuclear polarization (DNP) probe (1) suitable for performing high-resolution nuclear magnetic resonance (NMR) on liquid samples comprises: a corrugated waveguide (3) that transmits microwaves in the sub-millimeter wave range; four microwave reflectors (4, 5, 6, 7) that further transmit the microwaves to a sample (14) and are designed to spread the elongated microwave beam over an accessible sample area; and a cooling device (8, 9, 10) for avoiding heating the sample and maintaining its temperature at a specific level. The sample (14) is confined between two concentric quartz (QZ) tubes (15, 16) to form a thin, cylindrical layer that is smaller than the penetration depth of the excitation microwaves. This arrangement is called a sample assembly (13); it is surrounded by NMR coils (11, 12) and has the ability to rotate to increase the sample area irradiated by the microwaves and reduce the heating effect. In addition, the NMR coils (12, 13) are arranged to allow efficient transmission of microwaves through the sample assembly (13).

[0091] Label list

[0092] 1Probe assembly

[0093] 2 corrugated inverted cone

[0094] 3 Corrugated waveguide components

[0095] 4 Microwave reflectors

[0096] 5 Microwave reflectors

[0097] 6 Microwave reflectors

[0098] 7 Microwave reflectors

[0099] 8N2 gas flow Dewar tube

[0100] 9 Sample chamber Dewar flask

[0101] 10 Sample chamber exhaust cup

[0102] 11. First RF coil

[0103] 12 Second RF coil

[0104] 13 sample components (including 14, 15, 16)

[0105] 14 liquid samples

[0106] 15 external sample QZ tube

[0107] 16 inner sample QZ tube

[0108] 17 inner shield QZ tube

[0109] 18 external shielded QZ tube

[0110] 19N2 gas temperature sensor / heater port

[0111] 20N2 gas inlet (sample chamber)

[0112] 21 Teflon window

[0113] 22 connecting flange

[0114] 23 connecting flange

[0115] 24 probe support

[0116] 25N2 gas inlet (probe)

[0117] 26 Insulated sample chambers

[0118] 27 probe housing

[0119] List of prior art citations:

[0120] The following disclosures have been considered to assess the patentability of the present invention:

[0121] [1]Overhauser,AW,Polarization ofNucleiin Metals.Physical Review,1953.92(2):p.411-415.

[0122] [2]Hausser, D. and D. Stehlik, Dynamic Nuclear Polarization in Liquids. Advances in Magnetic Resonance, 1968.3: p.79-139.

[0123] [3] Bennati, M. and T. Orlando, Overhauser DNP in Liquids on C-13Nuclei. Emagres, 2019.8(1):p.11-18.

[0124] [4]Liu, GQ, et al., One-thousand-fold enhancement of high field liquidnuclear magnetic resonance signals at room temperature. Nature Chemistry, 2017.9(7):p.676-680.

[0125] [5]Dai,D.H.,et al.,Room-temperature dynamic nuclear polarizationenhanced NMR spectroscopy of small biological molecules in water.NatureCommunications,2021.12(1).

[0126] [6]Dubroca,T.,et al.,Large volume liquid state scalar Overhauserdynamic nuclear polarization at high magnetic field.Physical ChemistryChemical Physics,2019.21(38):p.21200-21204.

[0127] [7]Orlando,T.,et al.,Dynamic Nuclear Polarization of C-13Nuclei inthe Liquid State over a 10 Tesla Field Range.Angewandte Chemie-InternationalEdition,2019.58(5):p.1402-1406.

[0128] [8]Levien,M.,et al.,Nitroxide Derivatives for Dynamic NuclearPolarization in Liquids:The Role of Rotational Diffusion.Journal of PhysicalChemistry Letters,2020.11(5):p.1629-1635.

[0129] [9]Annino,G.,et al.,Magnetic resonance hyperpolarization and multipleirradiation probe head.2016,US9448290B2.

[0130]

[10] Prisner,T.and V.Denysenkov,Double-resonance structure and methodfor investigating samples by DNP and / or ENDOR.2013,US8570033B2.

[0131]

[11] Yoon,D.,et al.,High-Field Liquid-State Dynamic NuclearPolarization in Microliter Samples.Analytical Chemistry,2018.90(9):p.5620-5626.

[0132]

[12] Denysenkov,V.,D.H.Dai,and T.F.Prisner,A triple resonance(e,H-1,C-13)probehead for liquid-state DNP experiments at 9.4 Tesla.Journal ofMagnetic Resonance,2022.337.

[0133]

[13] Denysenkov,V.and T.Prisner,Liquid state Dynamic NuclearPolarization probe with Fabry-Perot resonator at 9.2 T.Journal of MagneticResonance,2012.217:p.1-5.

[0134]

[14] Nevzorov,A.A.,et al.,Characterization of photonic band resonatorsfor DNP NMR of thin film samples at 7 Tmagnetic field.Journal of MagneticResonance,2021.323.

[0135]

[15] Soundararajan,M.,et al.,Proton-detected solution-state NMR at14.1 T based on scalar-driven 13C Overhauser dynamic nuclearpolarization.Journal of Magnetic Resonance,2022.343.

[0136]

[16] Neugebauer,P.,et al.,Liquid state DNP of water at 9.2 T:anexperimental access to saturation.Physical Chemistry Chemical Physics,2013.15(16):p.6049-6056.

[0137]

[17] Nevzorov,A.A.,et al.,Multi-resonant photonic band-gap / saddle coilDNP probehead for static solid state NMR of microliter volume samples.Journalof Magnetic Resonance,2018.297:p.113-123.

Claims

1. An NMR probe (1) configured to perform high-resolution, liquid-state DNP-NMR and comprising: - a corrugated waveguide (3) for MW (= microwave) transmission arranged along the longitudinal axis Z of the probe (1), - a system of at least two MW mirrors (4, 5, 6, 7) for MW beam transmission, focusing and shaping to match the sample geometry, - at least one RF coil (11, 12) for NMR detection, mounted to allow the passage of mw to the sample region, It is characterized by - a cooling system comprising a flow dewar tube (8) mounted at the side of the probe and parallel to the corrugated waveguide (3) for feeding a cryogenic fluid and an insulated sample chamber (26) surrounded by a chamber dewar (9), - a sample rotation device configured to achieve uniform illumination over the sample volume to increase the sample volume under MW illumination and to reduce sample heating, - A sample assembly (13) comprising concentric circular tubes or rods (15, 16) forming a constraint for the sample layer (14).

2. The NMR probe according to claim 1, characterized in that The thickness of the sample layer (14) is between 0,25Δ and 1,25Δ, where Δ is the penetration depth of mw in the liquid sample medium.

3. The NMR probe according to any one of the preceding claims, characterized in that The sample assembly (13) comprises two concentric circular tubes or rods (15, 16).

4. The NMR probe according to claim 3, characterized in that The sample assembly (13) is made of a material with low mw absorption, preferably quartz or other mw transparent materials.

5. The NMR probe according to any one of the preceding claims, characterized in that Four MW mirrors (4, 5, 6, 7) are provided which transmit MW to the sample assembly (13) while maintaining proper B1 magnetic field polarization and forming a beam profile that is optimally distributed over the sample geometry to reduce its heating and increase the efficiency of MW excitation.

6. The NMR probe according to any one of the preceding claims, characterized in that The mirror (4-7) has a curvature based on a conical section, and in particular the mirror (4-7) is shaped as a quasi-ellipse.

7. The NMR probe according to any one of the preceding claims, characterized in that The system of MW mirrors (4, 5, 6, 7) for MW beam transmission, focusing and shaping comprises four mirrors (4, 5, 6, 7) positioned at distances of 17.25 mm, 42.5 mm and 34.50 mm from the sample axis (measured at the height of the coil center).

8. The NMR probe according to any one of the preceding claims, characterized in that Mirror (4, 5, 6, 7) by The sample is made of ceramic that is specifically coated with gold to minimize magnetic field distortion around the sample and maintain a high homogeneity of the static magnetic field B0.

9. The NMR probe according to claim 8, characterized in that The coating of the mirrors (4, 5, 6, 7) has a thickness between 3δ and 7δ, where δ is the skin depth in gold at room temperature (~0.15μm), specifically to maintain high conductivity of the skin layer for mW frequencies close to 263GHz, but still minimize magnetic field distortion around the sample.

10. The NMR probe according to any one of the preceding claims, characterized in that The mirrors (4, 5, 6, 7) are designed to transmit a MW beam (up to THz) with an appropriate B1 magnetic field polarization orthogonal to the static magnetic field B0 to saturate the ESR transition of the DNP, as well as to minimize the magnetic field distortion around the sample and maintain a high uniformity of the static magnetic field B0, which is required for high-resolution NMR.

11. The NMR probe according to any one of the preceding claims, characterized in that Two RF coils (11, 12) are provided, one of which is matched for a second resonant frequency to measure other nuclei or for 2 H lock.

12. The NMR probe according to any one of the preceding claims, characterized in that At least one shielding tube (17, 18) is concentrically arranged around the sample assembly (13) to guide the cryogenic fluid.

13. The NMR probe according to any one of the preceding claims, characterized in that The corrugated waveguide (3) is designed to minimize MW losses in the desired frequency range, maintain the desired polarization of the microwave magnetic field B1, and minimize the B0 magnetic field distortion around the sample.

14. The NMR probe according to any one of the preceding claims, characterized in that The cooling system comprises a device for controlling the temperature of the sample so as to minimize heating of the sample during mw excitation and to stabilize the temperature of the sample at a specific value.

15. The NMR probe according to any of the preceding claims, designed for high-resolution 1D and 2D DNP-enhanced NMR spectroscopy of magnetic nuclei exhibiting the Overhauser effect using radical polarizers, in particular using a non-resonant microwave setup.

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