Electron spin resonance microscope for imaging with micron resolution

What is claimed is:

1. An electron spin resonance (ESR) instrument for producing a magnetic resonance image of a specimen comprising: a miniature gradient coil; and a miniature microwave resonator operably connected to the miniature gradient coil, wherein: the magnetic resonance image is an electron spin resonance image, the electron spin resonance image has a resolution of at least 10.times.10.times.30 microns, the electron spin resonance image is acquired in a time period of less than 10 minutes, and the miniature microwave resonator applies an RF magnetic field for electron spin excitation and receives a resulting ESR signal.

2. The electron spin resonance instrument of claim 1, wherein said electron spin resonance image has a resolution of 5.times.5.times.10 microns.

3. The electron spin resonance instrument of claim 1, wherein said electron spin resonance image has a resolution of 3.times.3.times.8 microns.

4. The electron spin resonance instrument of claim 1, wherein said electron spin resonance image has a resolution of 1.times.1.times.5 microns.

5. The electron spin resonance instrument of claim 1, wherein said electron spin resonance image has an electron spin sensitivity of 10.sup.6 spins per voxel for 60 min acquisition at room temperature.

6. The electron spin resonance instrument of claim 1, wherein said electron spin resonance image has an electron spin sensitivity of 10.sup.5 spins per voxel for 60 min acquisition at room temperature.

7. The electron spin resonance instrument of claim 1, wherein said instrument operates at a frequency in the range of 9 to 60 GHz.

8. The electron spin resonance instrument of claim 1, wherein said electron spin resonance is continuous-wave electron spin resonance (CW ESR).

9. The electron spin resonance instrument of claim 8, comprising: (a) a conventional CW ESR spectrometer; (b) an imaging probe comprising a microwave resonator, and one or more gradient coils in electrical communication with said CW ESR spectrometer; (c) a signal conditioner for receiving, amplifying and conditioning said signal from said ESR spectrometer and providing an amplified and conditioned signal as output; (d) a computer which controls an imaging process and processes said conditioned output signal; and (e) at least one current driver for the gradient coils, said at least one current driver being controlled during said imaging process and driving at least one of said gradient coils.

10. The electron spin resonance instrument of claim 9, wherein said signal conditioner is a filter and a base band amplifier.

11. The electron spin resonance instrument of claim 10, wherein said baseband amplifier operates up to a frequency of 250 kHz.

12. The electron spin resonance instrument of claim 9, further comprising a mechanical fixture for holding a sample.

13. The electron spin resonance instrument of claim 9, further comprising: (f) a control unit.

14. The electron spin resonance instrument of claim 1, wherein said electron spin resonance is pulsed electron spin resonance.

15. The electron spin resonance instrument of claim 14, comprising: (a) a computer which controls the overall image acquisition process through a user interface; (b) a timing system; (c) a digitizer; (d) an analog output device; (e) a microwave reference source; (f) a low power pulsed microwave bridge; (g) a low power microwave transceiver; (h) a solid-state power amplifier; (j) at least one pair of gradient coil drivers; (i) at least one power source for driving said at least one pair of gradient coil drivers; (k) an imaging probe; (l) a high voltage tracking power supply; and (m) a monitor scope.

16. The electron spin resonance instrument of claim 15, wherein said computer is a personal computer.

17. The electron spin resonance instrument of claim 15, wherein said user interface is a graphical user interface.

18. The electron spin resonance instrument of claim 15, wherein said timing system comprises a plurality of TTL outputs, time resolution of less than or equal to 10 ns, programming time of 10 .mu.s or less, and minimal pulse length of less than or equal to 50 ns.

19. The electron spin resonance instrument of claim 15, wherein said analog output system comprises at least four analog outputs and an update rate of at least 200 kHz.

20. The electron spin resonance instrument of claim 15, wherein said microwave reference source comprises a power output of approximately 10 dBm through the 2-18 GHz range.

21. The electron spin resonance instrument of claim 15, wherein said low power microwave transceiver operates in the 6-17 GHz range.

22. The electron spin resonance instrument of claim 15, wherein said low power microwave transceiver is a homodyne transceiver that comprises one transmission channel with bi-phase modulation, which controls the individual pulse phase.

23. The electron spin resonance instrument of claim 22, wherein said individual pulse phase is a selected one of 0.degree., 90.degree., 180.degree., and 270.degree..

24. The electron spin resonance instrument of claim 1 further comprising a signal conditioner.

25. The electron spin resonance instrument of claim 24 further comprising means for coupling the miniature microwave resonator to the signal conditioner.

26. The electron spin resonance instrument of claim 1 further comprising a microwave bridge.

27. The electron spin resonance instrument of claim 26 wherein the microwave bridge is a CW ESR spectrometer.

28. The electron spin resonance instrument of claim 26 wherein the microwave bridge is a pulsed microwave bridge.

29. The electron spin resonance instrument of claim 1 wherein the RF magnetic field is a microwave magnetic field.

30. A miniature imaging probe for an electron spin resonance imaging apparatus, comprising a miniature microwave resonator and at least one gradient coil, wherein: the miniature microwave resonator applies an RF magnetic field for electron spin excitation and receives a resulting ESR signal, and the gradient coil is compatible with ESR operation at a frequency in the range of 9 to 60 GHz.

31. The miniature imaging probe for an electron spin resonance imaging apparatus of claim 30, wherein said miniature microwave resonator comprises high permittivity material.

32. The miniature imaging probe for an electron spin resonance imaging apparatus of claim 31, wherein said high permittivity material has a non-conducting dielectric ring structure.

33. The miniature imaging probe for an electron spin resonance imaging apparatus of claim 30, wherein said miniature microwave resonator is temperature stabilized by gas flow.

34. The miniature imaging probe for an electron spin resonance imaging apparatus of claim 30, further comprising a selected one of a microstrip, a dielectric antenna, and a coaxial antenna to obtain a high coupling coefficient.

35. The miniature imaging probe for an electron spin resonance imaging apparatus of claim 30, wherein said at least one gradient coil exhibits fast response to driving-current pulses when operated in pulsed mode.

36. The miniature imaging probe for an electron spin resonance imaging apparatus of claim 30, wherein said at least one gradient driver and coil minimizes power dissipation when operated in constant-gradient (CW-emulation) mode.

37. The miniature imaging probe for an electron spin resonance imaging apparatus of claim 36, wherein said at least one gradient coil consumes less than 1 Ampere when operated in constant-gradient (CW-emulation) mode.

38. The miniature imaging probe for an electron spin resonance imaging apparatus of claim 30, further comprising a shield material having electromagnetic shielding properties in the microwave regime, wherein the shield material provides electromagnetic transparency in the megahertz regime.

39. The miniature imaging probe for an electron spin resonance imaging apparatus of claim 30, wherein the miniature microwave resonator facilitates detection of an ESR signal.

40. The miniature imaging probe for an electron spin resonance imaging apparatus of claim 30 wherein the RF magnetic field is a microwave magnetic field.

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FIELD OF THE INVENTION

The invention relates to magnetic resonance imaging in general and particularly to high resolution electron spin resonance (ESR) imaging.

BACKGROUND OF THE INVENTION

Many fields of science and medicine require the close observation of small samples, for example, thin tissue slices extracted from living organisms or living/fixed cell cultures. Currently, the leading modalities for these kinds of observations are optical and fluorescence microscopy. These techniques are very mature and provide rich information regarding the investigated sample. Nevertheless, such optical-based modalities lack the ability to observe moderately thick three-dimensional (3D) non-transparent samples. They can not measure vital parameters such as molecular self diffusion and 3D flow vectors, and, for example in terms of medical tissues, many times result in inconclusive clinical diagnosis of histological samples. Furthermore, using optical methods, the following difficulties are encountered: it is difficult to image accurately the O.sub.2 partial pressure in specimens; it is difficult to recognize with high specificity various superoxides in the imaged sample; one can not measure a variety of image contrasts such as the spatially-resolved magnetic resonance relaxation times (T.sub.1 and T.sub.2) and the lineshape of the spins in the sample; and optical methods lack the possibility to correlate in-vitro with in-vivo measurements.

Nuclear Magnetic Resonance (NMR) microscopy, in which the nuclear spins can be considered as a unique kind of magnetic stain or dye, is currently a relatively widespread complimentary imaging tool for small samples that is found useful in many diverse clinical situations to achieve high identification specificity and measurement accuracy in cases of inflammation, fluid diffusion, blood flow and perfusion, lipid content, tissue types such as cancer, and tissue necrosis. NMR microscopy has also been found useful in materials science and botany to investigate and measure flow and porosity. Several NMR devices and applications of NMR microscopy are described in U.S. Pat. Nos. 5,258,710, 5,394,088, and 5,416,414. Thus, NMR microscopes are routinely employed in many fields of science and medicine, and several companies are producing such instruments commercially. Some people have even combined NMR and optical microscopes. The main drawback of the existing NMR microscopes is their high price (in the range of $500,000 to $1,500,000, mainly due to the superconducting magnet technology required for their operation), and the limited image resolution they offer (>10 microns), which can not rival the <0.5 micron resolution of optical imaging modalities. Due to these limitations (and regardless of the many potential advantages), the full potential of magnetic resonance microscopy cannot be readily exploited and such instruments are presently much less abundant than the optical-based microscopes in scientific and medical laboratories around the world.

Another, less common technique of magnetic resonance imaging, employs electron spin resonance (ESR) in paramagnetic molecules, rather than the spins of hydrogen nuclei. Whereas the field of NMR microscopy is well developed, ESR microscopy is not. Nevertheless, ESR has inherently many potential virtues over NMR, which could make this a technique of choice for Magnetic Resonance (MR) microscopic applications. For example, the signal per spin is much higher than in NMR, diffusion does not limit the resolution in the short time scales (T.sub.2's.about..mu.s) of the ESR measurements, ESR micro-resonators detect with a quality factor (Q) of .about.1000 compared to the Q.about.10 of the NMR micro-coils, and the ESR lineshape is more sensitive to dynamic effects, leading to richer information. An additional factor is the low cost of electromagnets used in ESR as compared to the expensive superconducting magnets of NMR microscopes. Since most samples do not contain stable paramagnetic molecules, paramagnetic species (often in the form of stable organic radicals) must be added in a manner similar to that of adding contrast agents in NMR or dyes in optics. This is a standard procedure, especially for microscopy, which also offers the benefit of eliminating any concerns associated with a large undesirable background signal, (such as protons in NMR). An ESR microscope can provide similar spatially resolved sample parameters to those obtained by NMR measurements, (i.e. spin concentration, relaxation times T.sub.1, T.sub.2, and diffusion coefficient), which compliments the information obtained by conventional optical microscopy.

Up to now, most ESR imaging (ESRI) efforts in biological samples have been directed towards observation of large subjects and to determining the radical and oxygen concentration (by its effect on the radical line width). U.S. Pat. Nos. 5,502,386, 5,578,922, 5,678,548 and 5,865,746 describe some of these efforts. Such experiments, conducted in-vivo, employ low fields of .about.10 mT at low RF frequencies (which results in relatively low spin sensitivity), so that the RF energy will penetrate deeply into the relatively large biological object. Consequently, a typical voxel resolution in low frequency ESR experiments is ca. [2 mm].sup.3. Most low-field ESR imaging techniques are based on Continuous Wave (CW) detection where the image is obtained by applying static gradients in various directions with respect to the object, which is sometimes referred to as the back projection technique. However, utilization of a single pulse Free Induction Decay (FID) sequence in conjunction with pulsed and static gradients has been also explored.

Previous publications, including a paper from one the inventors, have described in the past an ESR resonator based on high permittivity KTaO.sub.3 and demonstrated its application in the field of ESR spectroscopy. Such high permittivity small resonator structures, however, were not employed in the past as a basis for a miniature ESR imaging probe (which includes a resonator and the imaging gradient coils).

Other emerging techniques of high resolution ESR imaging include the use of magnetic tips (Magnetic Resonance Force Microscopy), Hall detection, scanning-tunneling microscopy (STM-ESR), and miniature microwave scanning probe. Nevertheless all these methods can be employed only to a very limited extent when botanically and biologically-related or relatively thick samples are considered. Thus, for example, the detection by magnetic tips as described in U.S. Pat. No. 6,683,451 suffers from low 3D sensitivity, especially when the samples are thicker than a few microns. Furthermore, this technique requires extreme physical conditions (high vacuum and often low temperatures), and can be employed only after complicated sample preparation. The STM-ESR is a surface (two-dimensional, or 2D) technique capable of handling only solid state samples placed over a conductive surface and also required extreme physical conditions for successful operation. The Hall detection and the miniature microwave scanning probe methods also operates only on the surface, or slightly below it, and have not proven to be useful in micron resolution imaging.

A number of problems in nuclear magnetic resonance imaging have been observed, including the need for strong magnetic fields, requiring expensive superconducting magnets, and the limited spatial resolution of images that are obtained.

There is a need for magnetic resonance imaging systems and methods that provide high resolution 2D and 3D images, especially for thin and thick biologically-related samples, at modest cost, and in short (1-10 min) acquisition times.

SUMMARY OF THE INVENTION

The present invention is an ESR-based microscope, which may be employed to obtain micron resolution images of biological- and materials science-related samples. Conceptually, it combines the fields of NMR microcopy with the field of large scale in-vivo ESR. One novel element of the invention lies in the use of specially designed miniature microwave resonators along with integral miniature magnetic field gradient coils, which facilitate the combination of high sensitivity and high spatial resolution. The miniature gradient coils set are placed very close to the resonator without affecting its microwave performance. Other novel aspects are related to the use of fast pulsed field gradient, unique sample containers, the use of special stable free radical material as an imaging contrast medium, and the system architecture.

A publication, published by the inventors less than a year before the submission of co-pending U.S. provisional patent application Ser. No. 60/598,100 of which priority is claimed, describes the general theory of ESR microcopy, both for CW and pulse operation, in terms of image signal-to-noise-ratio (SNR) and image resolution. It also provides an example of a 2D CW ESR imaging probe, with some measured results.

In one aspect, the invention relates to a electron spin resonance instrument for obtaining electron spin resonance images of a specimen. The electron spin resonance images have a resolution of at least 10.times.10.times.30 microns. The images are acquired in a time period of less than 10 minutes.

In one embodiment, the electron spin resonance image has a resolution of 5.times.5.times.10 microns. In one embodiment, the electron spin resonance image has a resolution of 3.times.3.times.8 microns. In one embodiment, the electron spin resonance image has a resolution of 1.times.1.times.5 microns. In one embodiment, the electron spin resonance image has an electron spin sensitivity of 10.sup.6 spins per voxel for 60 min acquisition at room temperature. In one embodiment, the electron spin resonance image has an electron spin sensitivity of 10.sup.5 spins per voxel for 60 min acquisition at room temperature.

In one embodiment, the electron spin resonance instrument is configured to operate using continuous-wave electron spin resonance (CW ESR). In one embodiment, the electron spin resonance instrument of comprises (a) a conventional CW ESR spectrometer; (b) an imaging probe comprising a microwave resonator, and one or more gradient coils in electrical communication with the CW ESR spectrometer; (c) a signal conditioner for receiving, amplifying and conditioning the signal from the ESR spectrometer and provides an amplified and conditioned signal as output; (d) a computer which controls an imaging process and processes the conditioned output signal; and (e) at least one current driver for the gradient coils, the at least one current driver being controlled during the imaging process and driving at least one of the gradient coils. In one embodiment, the signal conditioner is a filter and a base band amplifier. In one embodiment, the baseband amplifier operates up to a frequency of 250 kHz.

In one embodiment, the electron spin resonance instrument further comprises a mechanical fixture for holding a sample. In one embodiment, the electron spin resonance instrument further comprises (f) a control unit.

In one embodiment, the magnetic resonance instrument is configured to operate using pulsed electron spin resonance. In one embodiment, the electron spin resonance instrument comprises (a) a computer which controls the overall image acquisition process through a user interface; (b) a timing system; (c) a digitizer; (d) an analog output device; (e) a microwave reference source; (f) a low power pulsed microwave bridge; (g) a low power microwave transceiver; (h) a solid-state power amplifier; (j) at least one pair of gradient coil drivers; (i) a pre-regulated high voltage power supply; (k) an imaging probe; (l) a high voltage tracking power supply; and (m) a monitor scope.

In one embodiment, the computer is a personal computer. In one embodiment, the user interface is a graphical user interface. In one embodiment, the timing system comprises a plurality of TTL outputs, time resolution of less than or equal to 10 ns, programming time of 10 .mu.s or less, and minimal pulse length of less than or equal to 50 ns. In one embodiment, the timing system comprises at least four analog outputs and an update rate of at least 200 kHz. In one embodiment, the microwave reference source comprises a power output of approximately 10 dBm at the 2-18 GHz range. In one embodiment, the low power microwave transceiver operates in the 6-17 GHz range. In one embodiment, the low power microwave transceiver is a homodyne transceiver that comprises one transmission channel with bi-phase modulation, which controls the individual pulse phase. In one embodiment, the individual pulse phase is a selected one of 0.degree., 90.degree., 180.degree., and 270.degree..

In another aspect, the invention features a miniature imaging probe for an electron spin resonance imaging apparatus, comprising a microwave resonator and at least one gradient coil.

In one embodiment, the microwave resonator comprises high permittivity material. In one embodiment, the high permittivity material has a non-conducting dielectric ring structure. In one embodiment, the microwave resonator is configured to permit gas flow for temperature stabilization of the microwave resonator. In one embodiment, the miniature imaging probe for an electron spin resonance imaging apparatus further comprises a microstrip to obtain a high coupling coefficient. In one embodiment, the at least one gradient coil is configured to exhibit fast response when operated in pulsed mode. In one embodiment, the at least one gradient coil is configured to consume less than 1 Ampere when operated in CW mode.

In one embodiment, the miniature imaging probe for an electron spin resonance imaging apparatus further comprises a shield material having electromagnetic shielding properties in the microwave regime, while providing electromagnetic transparency in the megahertz regime.

In one embodiment, the resonator material possesses at least one of the following unique features and advantages for ESR microscopy: a high concentration of electromagnetic [EM] field by high-permittivity dielectric (thereby yielding a high SNR and a small "effective volume" from which the signal is obtained); a proportional concentration of B.sub.1 field and correspondingly reduced r.f. (radio-frequency/microwave) input power; a proportional reduction of power dissipation in gradient and modulation coils; a smaller resonator and consequently reduction in the requirement for the volume of homogenous B.sub.0, which allows the use of a smaller primary magnet; a minimized resonator volume that alternately permits the use of a permanent magnet assembly (with vernier scan coils) for the B.sub.0 field; a non-conducting dielectric ring structure that is inherently immune to eddy current effects. In one embodiment, the construction features facilitate specimen insertion and manipulation (e.g., for precise positional adjustment of high permittivity bio-samples). In one embodiment, the open design permits unimpeded gas flow for temperature stabilization of resonator (for controlled-temperature experiments and removal of heat generated in gradient and modulation coils). In one embodiment, the microwave coupling scheme utilizes microstrip or thin coaxial antenna to obtain high coupling coefficient in limited space without introduction of a deleterious metallic ground plane in sensitive regions (which can cause the reduction of the resonator Q or enable Eddy currents that disturb the magnetic field near the sample). Coupling may also be achieved through the use of a dielectric waveguide antenna. Such an antenna allows the elimination of any metallic parts in the vicinity of the resonator and consequently provides better immunity to eddy currents effects that may arise due to the pulsed field gradients.

In one aspect, the invention features efficient magnetic field gradient coils for CW and pulsed imaging. In the CW embodiment, the coils consume currents of less than 1 A to produce the necessary gradients. In the pulse embodiment, the coils exhibit the fast response necessary for pulsed imaging.

In one aspect, the invention relates to shielding for the imaging probe that comprises a material having electromagnetic shielding properties in the microwave regime, while providing electromagnetic transparency in the megahertz spectral regime, of the applied pulsed field gradients.

In one aspect, the invention relates to the use of stable organic radicals for microscopic calibration and labeling applications. In some embodiments, the organic radicals include, for example, trityl ((tris-(8-carboxyl-2,2,6,6-tetrakis-methyl-benzo[1,2d:4,5-d']bis(1,3)dith- iole)methyl), LiPc (Lithium Phthalocyanine radical) and their derivates. In addition, functional and sample preparation aspects related to the invention such as pulsed 2D/3D ESR imaging, sample preparation methods, spectrometer architecture, and CW and pulse current drivers are believed to be novel and patentable.

Other exemplary features of the invention that are believed to be significant include: the possibility in CW mode of varying the phase and or frequency of the excitation of the gradient coils during the integration time to improve the SNR and/or to reduce artifacts; the use of high permittivity material with low losses as components of the imaging probe; the ability to position samples in a millimeter-sized resonator to accuracies of 50 to 100 microns (and proportionally more accurately as the resonator size is reduced); the use of automatically controlled or computer controlled (as compared to manually controlled) high gain amplifiers in baseband amplifier and filtering units; the possibility of using temperature shift at the resonator for tuning the resonance frequency; and the use of many generally known deconvolution methods, and associated computer programs or software modules for obtaining images in the projection reconstruction method.

In some embodiments, data obtained from the use of the ESR microscope may be combined with confocal optical and/or fluorescence microscopy data to provide better assignment of the image and more complete information about the imaged object.

Other types of samples that may be imaged by ESR microscopy are samples for materials science applications (such as paramagnetic semiconductors), samples for botanical applications, and even limbs or organs of small animals (ex-vivo and in-vivo).

In another aspect, the invention relates to the process of operation of an instrument used to obtain magnetic resonance images with a resolution of better than 1.times.1.times.5 microns in several minutes of acquisition for small samples.

The foregoing and other objects, aspects, features, and advantages of the invention will become more apparent from the following description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The objects and features of the invention can be better understood with reference to the drawings described below, and the claims. The drawings are not necessarily to scale, emphasis instead generally being placed upon illustrating the principles of the invention. In the drawings, like numerals are used to indicate like parts throughout the various views.

FIG. 1 shows a block diagram of an exemplary CW ESR microscope, according to principles of the