Electron spin resonance microscope for imaging with micron resolution Number:7,403,008 from the United States Patent and Trademark Office (PTO) owispatent ESR microscope systems and methods for examining specimens using both continuous wave and pulsed modes in the 9 to 60 GHz range. The ESR microscope uses an image probe comprising gradient coils in addition to conventional modulation coils (in continuous wave mode) or magnetic field bias coils (in pulse mode), and a resonator constructed from high permittivity material. The systems and methods also involves the use of sample containers that permit the precise placement of samples in relation to the image probe. The microscope uses a microstrip or thin coaxial or dielectric antenna to obtain a high coupling coefficient to the specimen being imaged. The microscope systems provide resolution at the single micron level, and permit the observation of images comprising tens to hundreds of pixels for each of two or three dimensions in a few minutes. Novel stable radicals used as the imaging media are also described.<H resolution, microscope, Electron, spin, r, 7403008.html, Patent, pto, 7403008

Title: Electron spin resonance microscope for imaging with micron resolution

Abstract: ESR microscope systems and methods for examining specimens using both continuous wave and pulsed modes in the 9 to 60 GHz range. The ESR microscope uses an image probe comprising gradient coils in addition to conventional modulation coils (in continuous wave mode) or magnetic field bias coils (in pulse mode), and a resonator constructed from high permittivity material. The systems and methods also involves the use of sample containers that permit the precise placement of samples in relation to the image probe. The microscope uses a microstrip or thin coaxial or dielectric antenna to obtain a high coupling coefficient to the specimen being imaged. The microscope systems provide resolution at the single micron level, and permit the observation of images comprising tens to hundreds of pixels for each of two or three dimensions in a few minutes. Novel stable radicals used as the imaging media are also described.

Patent Number: 7,403,008 Issued on 07/22/2008 to Blank, et al.

Inventors: Blank; Aharon (Ithaca, NY), Dunnam; Curt R. (Trumansburg, NY), Borbat; Peter P. (Ithaca, NY), Freed; Jack H. (Ithaca, NY)
Assignee: Cornell Research Foundation, Inc. (Ithaca, NY)

Appl. No.: 11/195,388

Filed: August 2, 2005

Related U.S. Patent Documents


Application Number	Filing Date	Patent Number	Issue Date
60598100	Aug., 2004

Current U.S. Class: 324/316

Current International Class: G01V 3/00 (20060101)

Field of Search: 324/300-322

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Foreign Patent Documents


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Other References

TA. Carpenter et al., "Magnetic Resonance Microscopy"; IEE, 1994 pp. 1-2. cited by examiner .
Blank A et al.: "High resolution electron spin resonance microscopy" Journal of Magnetic Resonance, Academic Press, Orlando, FL US, vol. 165, No. 1, Nov. 2003, pp. 116-127, XP004466389 ISSN: 1090-7807 abstract p. 116, col. 1,2-p. 125, col. 1, 2. cited by other .
Quine R. W. et al.: "Pulsed EPR Spectrometer" Review of Scientific Instruments, AIP, Melville, NY, US vol. 58, No. 9, Sep. 1, 1987 pp. 1709-1723, XP000605540 ISSN: 0034-6748; p. 1709-p. 1721. cited by other.

Primary Examiner: Shrivastav; Brij B.
Assistant Examiner: Vargas; Dixomara
Attorney, Agent or Firm: Marjama Muldoon Blasiak & Sullivan LLP

Government Interests

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support from the National Science Foundation (NSF) under Grant No. CHE0098022. The Government has certain rights in the invention.

Parent Case Text

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of co-pending U.S. provisional patent application Ser. No. 60/598,100, filed Aug. 2, 2004, which application is incorporated herein by reference in its entirety.

Claims

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.

Description

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 invention.

FIG. 1A shows a block diagram of an alternative embodiment of a CW ESR microscope, according to principles of the invention.

FIG. 2 shows a block diagram of an exemplary pulsed ESR microscope, according to principles of the invention.

FIGS. 3a, 3b, and 3c show schematic drawings of an exemplary imaging probe, according to principles of the invention.

FIG. 3d is an exploded view of an embodiment of three orthogonal gradient coil pairs, according to principles of the invention.

FIG. 4 shows an exemplary sample container, according to principles of the invention.

FIG. 5 shows an exemplary imaging sequence for pulsed ESR imaging, according to principles of the invention.

FIG. 6 shows an example of a CW ESR image of a solid test sample, according to principles of the invention.

FIGS. 7a-7f show 2D and 3D ESR images acquired using apparatus and methods embodying principles of the invention, and a corresponding optical image.

FIG. 8a is an optical image of a LiNc-BuO liquid suspension in a thin nylon mesh.

FIG. 8b is an ESR image of the LiNc-BuO liquid suspension obtained according to principles of the invention.

FIG. 9 shows an example of a pulsed ESR image of a liquid sample typical of a biological specimen, and a corresponding optical image, according to principles of the invention.

DETAILED DESCRIPTION OF THE INVENTION

An Electron-Spin Resonance [ESR] (also referred to as Electron-Paramagnetic Resonance [EPR]) microscope is described, and methods of use of the microscope are presented. This imaging system overcomes the resolution limitations of existing NMR-based magnetic resonance microscopy and provides complimentary 3D information to optical microscopy, in a sub-micron resolution. The ESR microscope may be realized as a compact stand-alone instrument or as a retrofit compatible with existing ESR spectrometer instruments. Two distinct means of obtaining ESR micro-images, based on continuous-wave [CW] and pulsed signal acquisition methods, are possible with minor variation of the ESR microscope invention. As used in this application, the term "image" and its variants (e.g., images, imaging, and so forth, whether used as a noun or as a verb) refer to spatially resolved (1D, 2D, or 3D) information that can be obtained about the examined sample, or the act of obtaining such information, using the inventive instruments and methodologies described herein. This information can be any of the spin concentration, the spin relaxation times (T.sub.1, T.sub.2), the line shape of the spins, the diffusion coefficient of the spin probes, or any other spatially resolved parameters known in the art to be measurable by magnetic resonance imaging techniques.

We describe new instruments and methodology to obtain high resolution (better than 1 micron) magnetic resonance images of small samples (mainly, but not limited to, biological samples) with relatively simple and inexpensive means. The method relies on the imaging of stable radicals, which are affixed to the sample, rather than observing the hydrogen protons or other nuclear spins (as NMR does). The imaging of stable radicals (termed Electron Spin Resonance, or ESR, imaging) is a well known technique commonly employed in the scientific community for more than two decades. It provides similar or, in same cases, even superior information to that obtained by NMR imaging. The process of affixing the stable radicals to the sample is fairly well developed and is similar to applying contrast agents in NMR or fluorescence probes/dyes in optical microscopy. However, the previous resolution obtained by ESR imaging (especially for bio-samples) has been limited to .about.0.1 mm for 2D and 3D images. We overcome this limitation and can produce resolution that is better than 1 micron, for a relatively short acquisition time of several minutes, by the combination of a new microwave resonator, efficient gradient coils, advanced imaging algorithms, novel stable radicals used as the imaging media, the use of higher microwave frequencies, and data acquisition technology. Combining these novel subsystems enables us to produce a desktop ESR microscope, based on electromagnet and modern microwave technology that facilitates fast, low cost and reliable magnetic resonance imaging of small samples for a variety of biological, biomedical and materials science applications.

General System Description

The ESR microscope is comprised of a static magnetic field source, a microwave source and a detector, a microwave resonator, a mechanism for sample positioning and alignment, at least one and preferably three gradient coils, optionally bias coils for Field Frequency Lock (FFL) (a mechanism that uses the ESR signal to correct for any magnetic field drifts by means of DC biasing one of the gradient coils within the imaging probe), and a computer for control and data processing. Either continuous-wave or pulsed operational ESR microscopy instruments are possible with appropriate sub-system variations, and have been demonstrated. In the ESR microscope, a primary magnetic field polarizes the electron spins of the free radicals. The gradient coils generate magnetic fields that are added to the static main field and vary across the sample volume to facilitate the spatial resolution of the system. The microwave source excites the spins, producing characteristic signals, which are concentrated by the microwave resonator, then detected, digitized, and analyzed in the computer.

System Features

In the embodiments described herein, the high resolution of the ESR microscope is achieved through the use of one or more of the following system features: 1. a miniature resonator (typical size of 0.3-3 mm, depending on the frequency); 2. efficient gradient coils producing gradients of .about.1-10 Tesla per meter (T/m) for one ampere (1 A) of current (for CW and pulsed systems); 3. the ability to apply either or both of integrated modulated-gradient field [MGF] and projection reconstruction [PR] methods for CW imaging; 4. advanced fast pulsed magnetic field gradient current drivers; and 5. high repetition averaging capability for pulsed-mode imaging.

System Capabilities

The systems of the invention described herein have been demonstrated on samples having size: 4.times.4 mm and .about.0.3 mm thick. It is believed that a resolution of better than 1.times.1.times.5 micron can be attained (depending on frequency of operation). To date, in some embodiments, .about.5.times.5.times.10 micron resolution has been attained in pulse mode at 9 GHz, .about.3.times.3.times.8 micron resolution has been attained in pulse mode at 16 GHz, and .about.10.times.10.times.30 micron resolution has been attained in CW mode at 9 GHz. Typical image acquisition time is approximately 10 min for a 256.times.256.times.64 pixel (or voxel) image in pulse mode and .about.60 min for 128.times.128.times.32 pixel images in CW mode. The high resolution results (down to sub micron) mentioned should be achieved at higher frequencies, e.g. 35-60 GHz. Different types of images representing different type of information from the same sample, may be obtained through the use of different pulse sequences as known in the art (e.g., image of the radical concentration, image of the lineshape of the radicals, image of the spatially resolved relaxation parameters T.sub.1, T.sub.2 of the radicals, or the self diffusion coefficients through the use of fixed or pulsed field gradients). It is expected that improved manufacturability or performance will be achieved with further modifications of the apparatus. 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. It is believed that the electron spin resonance image can have an electron spin sensitivity of 10.sup.5 spins per voxel for 60 min acquisition at room temperature.

Detailed Hardware Description

CW ESR Microscope

FIG. 1 shows a block diagram of an exemplary CW ESR microscope 100. The CW ESR microscope instrument comprises: (a) a conventional CW ESR spectrometer 105, for example a commercially available CW ESR spectrometer such as a Varian, Bruker, or JEOL instrument, modified with: (b) an imaging probe 110 that includes a microwave resonator, a mechanical fixture for holding the sample, one or more gradient coils (preferably for three orthogonal axes), and a magnetic field modulation coil (which is similar to a CW ESR resonator); (c) a dedicated computer 115 which controls the imaging process and acquires the ESR signal; (d) at least one current driver 120 for the gradient coils (and preferably three current drivers for driving three gradient coils); and (e) a baseband (up to .about.250 kHz) amplifier and filter unit (signal conditioner 125) which obtains the raw ESR signal from the CW ESR spectrometer and preconditions it to enable the signal to be digitized, for example by an A/D card. The computer can be any commercially available general purpose programmable computer having sufficient computational power to perform the required computations. The computer can be, for example, a desktop computer, a laptop computer, or another commercially available computation device having memory for holding programmed instructions and data, and input and output ports and devices for accepting instructions from a user and for reporting information to the user in any convenient form. The computer can run data acquisition software (for example LabView version 7 software available from National Instruments, or another user interface comprising a graphical user interface (GUI)), and comprises an analog-to-digital (A/D) card 130 (for example, the PCI-6023 from National Instruments) for data digitization and acquisition. The computer also comprises a digital-to-analog (D/A) card 135 (for example, a PCI analog output card with 8 outputs and an update rate of up to .about.200 kHz, such as the PCI-6713 from National Instruments) to generate analog output signals, such as the gradient waveforms for the three imaging axes and the waveform for the modulation coil. The current waveform in the gradient coils can be generated either directly at the computer analog output card or in a control unit as described in the alternative embodiment hereinbelow.

In operation, a sample is loaded into the imaging probe 110. The CW ESR spectrometer 100 applies electromagnetic energy to the imaging probe 110 and receives return signals. The return signals are communicated to the ESR signal conditioner 125 and then passed to the A/D 130 for digitization. The digital signals produced are communicated to the computer 115, which is operating software to analyze the digital signals and to display results to a user. The computer 115 can also communicate control signals to the imaging probe 110 by way of D/A 135 that converts digital signals generated by the computer 115 to analog signals that are communicated to the current drivers 120 which control the signals applied to gradient coils operating in conjunction with the imaging probe. The gradient coils apply gradient signals to the imaging probe 110, thereby spatially encoding the signal detected by the CW ESR spectrometer 105, which spatially encoded signals are then fed back to the computer 115 as previously described. A user or a programmed sequence can command the computer 115 to modify the gradients according to principles that will allow the discovery of useful ESR information about the sample of interest.

FIG. 1A shows a block diagram of an alternative embodiment of a CW ESR microscope, according to principles of the invention. The second embodiment of the CW ESR microscope has the following components: a. conventional CW ESR spectrometer; b. dedicated computer which controls the imaging, process and acquires the ESR signal; c. current drivers for the gradient coils; d. baseband (up to .about.250 kHz) amplifier and filter unit (signal conditioner); e. control unit; and f. imaging probe that includes the microwave resonator, mechanical fixture for holding the sample, and gradient coils.

To image a sample, one must first attach the imaging probe (described in greater detail with respect to FIG. 3a-3d hereinbelow) to the CW ESR spectrometer (by replacing the conventional resonator). The imaging probe is connected to the spectrometer through the microwave (MW) input/output of the spectrometer and the modulation coils line (similar to any CW-ESR resonator). In addition, the probe is connected to the gradient current drivers and to air and water cooling lines. The water and air cooling lines are useful to maintain a stable resonance frequency of the high permittivity resonator (FIG. 3b). The investigated sample should be sealed (under normal or deoxygenated atmosphere) between two flat glass cover slips and inserted, with the help of the sample holder, to a well-defined, closely controlled position in the center of the resonator. Following this, the ESR spectrometer is set to acquire the signal of the sample at the required conditions of MW power, static magnetic field, and main modulation. After fixing the spectrometer on the maximum of the ESR signal, the computer controlled imaging procedure is initiated, and the gradient coils are activated to obtain the image. In some embodiments, the ESR microscope incorporates a Field Frequency Lock (FFL) system that adjusts the main (static) magnetic field by biasing the modulation coils of the imaging probe, and maintains the on-resonance condition throughout the period of the imaging experiment. The FFL mechanism uses the ESR signal to correct for any magnetic field drifts by means of a bias coil within the imaging probe that determines the local static field at the sample (large electromagnet field+bias coil field). Thus, the basic commercial CW ESR spectrometer is completely "unaware" of the imaging procedure, with the interfaces between the spectrometer and the imaging probe/system being kept to a minimum. The image is displayed on a monitor connected to the computer in real time during the acquisition process and then can be saved and/or manipulated as necessary.

CW ESR spectrometer. Most commercial CW ESR spectrometers (e.g. from Bruker, Varian, or JEOL) can be used as a basis for the imaging module. The spectrometer serves as a stable, amplitude controlled, MW source that is independently frequency locked on the resonance frequency of the imaging probe, by means of the spectrometer Automatic Frequency Control (AFC). The spectrometer also provides a current drive for the regular modulation coils of the imaging probe and should allow for the operator to fix the external static magnetic field close to the resonance field of the imaged sample. The MW ESR signal returning from the imaging probe is detected by the spectrometer and is pre-amplified (and otherwise conditioned) at the spectrometer MW bridge. In the case of a spectrometer (Varian E-12), we inserted prior to the diode detection of the ESR signal from the resonator a low noise X-band pre-amplifier (Miteq AFS3-08001200-14-ULN). This amplifier improves the SNR of the spectrometer by a factor of .about.5 and its amplification gain (.about.25 dB) enables the AFC of the Varian bridge to lock on the returning signal even for low MW power (.about.1 .mu.W), a power level that is common in the present imaging technique. The diode detected baseband signal is directly fed from the bridge pre-amplifier (similar to the case of Time Resolved ESR measurements) to a signal conditioning unit and then goes to the PC for sampling and further analysis.

Control computer and imaging software: The entire imaging process is controlled by a computer, such as a standard personal computer (PC), equipped with an analog input+digital I/O card (for example, National Instruments 6023E) and an analog output card (for example, National Instruments 6713). These cards enable arbitrary waveform generation and fast sampling of signals up to several hundred kHz. The digital analysis of the sampled diode detected ESR signal supersedes the need to employ a conventional lock-in amplifier while simultaneously obtaining all the ESR signal harmonics, in the correct phase, with respect to the main modulation current. In one embodiment, the software operating on the system is capable of acquiring 2D images at any given location (using 3D slice selection), 3D images, and 4D spatially resolved ESR line shape (spectral-spatial projection reconstruction imaging). The imaging methods that can be employed are either the PR or the MFG method. Both methods can in principle employ the MFG method for Z slice selection. The control software (based on LabView) obtains the imaging parameters from the user. These parameters include, for example, the number of pixels in the image (along the x and y axes), the current amplitude in the gradient coils, the waveform and frequency used in the modulated gradient coils (e.g., sine, serrasoid, etc.), the image extent in mm, and parameters related to the functionality of the FFL system. The software can also acquire the normal ESR signal (first and second harmonics), by sweeping the magnetic main (static) field with the probe's gradient coils. At the end of the imaging process the data can be saved and/or further processed with the aid of Matlab software or other similar mathematical manipulation software. Such post processing includes, for example, in the case of image acquisition through the PR method, de-convolution of the projections and inverse Radon transformation to obtain the image.

Current drivers for the gradient coils: The gradient coils are driven by six programmable current sources, one current source for each coil, each capable of supplying up to 3 A of arbitrary waveform current, in the DC-10 kHz range. Each current driver is based on a simple feedback loop. In practice, the coils in the present system do not require more than 1 A to generate high enough gradient fields. The low current consumption of the system greatly simplifies the design and space requirements for the driver unit.

Baseband amplifier and filter unit (signal conditioner): In most ESR spectrometers, the ESR signal, detected by the MW diode, goes through a baseband preamplifier that is part of the commercial bridge. In some embodiments, the signal level after this pre-amp is not high enough to be sampled directly by the A/D card in the computer. To facilitate proper A/D sampling we employ a signal conditioning unit comprising band-pass filters and a high gain amplifier. The dual band-pass filter transfers only signal at the regular modulation frequency generated by the CW spectrometer, and its second harmonic. In one embodiment, there is provided a filter that transfers 25 kHz and 50 kHz and the spectrometer is operated at 25 kHz modulation frequency. The high gain amplifier (such as the Tektronix AM502) that follows the dual band-pass filter produces variable gain (which in some embodiments can be manually controlled) in the range of 40-100 dB.

Control unit: The current waveform in the gradient coils can be generated either directly at the computer analog output card or optionally in the control unit. If the computer analog output card is used, the control unit is optional. The control unit receives commands from the computer via the 8 bit digital Input/Output (I/O) bus of the 6023E card and subsequently synthesizes the required waveforms. The control unit is limited in the complexity of the waveform it can generate (as compared to the arbitrary waveform generator of the analog output card), but for most imaging tasks it has sufficient capabilities. Its usefulness is that it reduces the overhead time in the imaging procedure related to the calculation and generation of the different waveforms in the computer and thus shortens the actual acquisition time by a factor of .about.2. Computer systems operating a high enough clock speeds do not require the use of a control unit, but can perform all of the necessary calculations and control operations directly.

Imaging probe: The imaging probe is an important component of the CW imaging system. A schematic drawing of the imaging probe is given in FIG. 3a. The probe is based on a double stacked ring resonator, machined from SrTiO.sub.3 single crystal (Commercial Crystals, FL. USA). This crystal has permittivity of .about.300 at room temperature and tan .delta..about.5.times.10.sup.-4. Each one of the resonator rings is 0.55 mm in height with outer diameter of 2.31 mm and inner diameter of 0.81 mm. When the two rings were placed at a separation of .about.0.4 mm, the fundamental resonance frequency of this structure was found to be .about.9.15 GHz (in the center of the working frequency of the Varian E-12 ESR spectrometer). The resonator is excited by a microstrip transmission line on a low-loss substrate (GE electromaterials GTEK ML200D Epoxy/Polyphenylene Oxide resin). The excitation geometry and the calculated fields of this resonator at the resonance frequency have been calculated using CST Microwave Studio. The "effective volume" of the resonator, as derived from the calculated magnetic fields is .about.3 mm.sup.3 for a ring separation of 0.4 mm. The two high permittivity rings are held by a Delrin part at a fixed distance (see FIG. 3b). This Delrin spacer also holds the flat sample and enables precisely controllable positioning of the imaged object in the center of the resonator. Variable coupling is achieved by changing the distance between the resonator rings and the microstrip line using a linear 1D stage (for example, the non-magnetic model MDE 255 from Elliot Scientific, GB). In addition, the vertical position of the rings with respect to the microstrip line can be varied slightly by moving the Delrin part up and down and/or adjusting the vertical microstrip line position. This variability enables optimal control of the rings' coupling, for a wide variety of samples.

The resonator rings are surrounded by a thin (for example, 0.12 mm thick) hollowed brass cylinder that shields the resonator at the microwave frequencies, but is transparent to the low frequency magnetic field gradients. The regular modulation of the CW spectrometers (commonly at 25-100 kHz) penetrates through the shield opening along the cylinder axis. The loaded Q of the resonator was found to degrade slightly, from .about.1200 to .about.900, when inserting it into the brass shield. The gradient X, Y, and Z coils are arranged around the cylindrical shield, along with the regular modulation coils. The gradient coils are made from bondable 38 AWG copper magnet wire, and the modulation coils are made from bondable 32 AWG wire. The innermost coils are the Y and Z gradient coils respectively, which are based on a "Golay" design. The X gradient coils (for example, a standard Maxwell pair), are positioned in the outermost layer of the gradient coils, followed by the main modulation Helmholtz pair. Gradient efficiency is 4, 5, and 1.2 T/(m*A), and coil resistance is 9, 8.5, and 4.4 .OMEGA. for the X, Y, and Z coils respectively. These figures significantly improve upon an earlier 2D probe design, which achieved gradient efficiency of .about.1.5 and 2.5 T/(m*A), for the Y and X coils respectively, with coil resistance of 8 .OMEGA..

The probe structure is cooled by water flow, which maintains the entire brass structure at constant temperature. In addition, the rings themselves are cooled by air or He flow to maintain a stable resonance frequency, since the SrTiO.sub.3 is highly sensitive to temperature changes (at X-band the drift is .about.20 MHz per .degree. K.). In principle, the water/air flow system can be temperature controlled to regulate the temperature in the range (.about.0-50.degree. C.). The imaging probe can accommodate flat samples with dimensions of .about.1.5.times.1.5 mm (corresponding to the active area/volume of the probe) and a height of up to .about.0.5 mm (depending on the separation of the resonator rings). In practice, for liquid samples, one should contain the sample in a glass structure. It is convenient to employ thin cover slips with a small acid etched "well", as a convenient container for the liquid samples, as described hereinbelow (FIG. 4). Such a design enables one to measure a net sample size of .about.1.5.times.1.5 mm and a height of .about.0.25 mm. These samples can be sealed, if necessary, under Argon atmosphere, by the use of UV curable glue.

The spin sensitivity of the imaging probe was measured with a 1 mM deoxygenated water solution of protonated trityl radical. An SNR of 1.6.times.10.sup.4 was obtained for a 0.06 .mu.L sample, with 1 s time constant and .about.0.2 G field modulation. This SNR is in good agreement with the results of our recent prediction of an SNR of 1.8.times.10.sup.4 for such a trityl sample. The measured SNR corresponds to a spin sensitivity of 2.2.times.10.sup.9 spins/ Hz, and a concentration sensitivity of .about.6.2.times.10.sup.-8 M/ Hz, for the flat sample considered here. The combination of high spin and concentration sensitivities, for samples of small volume, is very helpful to facilitate high resolution imaging capability.

As noted above, the system can employ two different imaging methods to obtain the ESR image, within the framework of CW acquisition. Some of the results discussed herein were obtained with the PR method, which is a common method used to acquire in-vivo CW-ESR images. This tomographic imaging technique has been described in detail in many previous publications, and we shall not elaborate on it herein. Other results presented here were collected employing the MFG method. This method is less commonly employed since it is mainly advantageous for microscopic applications. The MFG method has been described previously, with discussions of the image acquisition technique, image Signal-to-Noise-Ratio (SNR), and gradient coil requirements. Nevertheless, we include here, for clarity, a short outline of this method, and also present a discussion of image resolution in 2D and 3D imaging, as a function of the modulated field gradient amplitude.

The idea behind the MFG method is to over-modulate the entire imaged sample, apart from a single voxel, from which the ESR signal is obtained. The over-modulation of the sample is achieved by a set of gradient coils excited by a low frequency periodic current. These coils have a null field point that can be swept in space by changing the relative current amplitude in each coil that produces the gradient field. Let us analyze more quantitatively the imaging scenario and obtain the image resolution for various cases. The time domain CW ESR signal in the case of conventional field modulation is given by (equation [1]): S(t, B.sub..delta.)=S.sub.0/{[.DELTA.B.sub.1/2/2].sup.2+(B.sub..delta.+B.sub.m sin .omega..sub.mt).sup.2} where .DELTA.B.sub.1/2 is the Full Width Half Maximum (FWHM) of the ESR line; B.sub..delta.=(B