WO2024256974A1

WO2024256974A1 - Systems and methods for spin-network labeled magnetic resonance imaging

Info

Publication number: WO2024256974A1
Application number: PCT/IB2024/055724
Authority: WO
Inventors: Ilai SCHWARTZ; Stephan Knecht
Original assignee: Nvision Imaging Technologies Gmbh
Priority date: 2023-06-12
Filing date: 2024-06-11
Publication date: 2024-12-19

Abstract

Systems and methods are presented for acquiring MRI images based on low-abundance molecules in a subject or a portion thereof. The acquired MRI images can then be used to determine a biological status of the subject or the portion thereof. The systems and methods generally utilize a target molecule containing at least two nuclear spins. When the subject or the portion thereof is placed in the magnetic field of an MRI machine, MRI-active molecules in the subject or the portion thereof generate magnetization. Target magnetization associated with the target molecule or a metabolic derivative thereof is selectively preserved utilizing the corresponding J-coupling network. Non-target magnetization associated with other MRI-active molecules is suppressed and nearly all of the MRI signal results from the target magnetization. The systems and methods may allow for the acquisition of MRI images based on a single low- abundance molecule, free of confounding background signals from higher-abundance molecules.

Description

SYSTEMS AND METHODS FOR SPIN-NETWORK LABELED

MAGNETIC RESONANCE IMAGING

CROSS-REFERENCE

[001] The present application claims priority to U.S. Provisional Patent Application No. 63/472,504, entitled “SYSTEMS AND METHODS FOR SPIN-NETWORK LABELED MAGNETIC RESONANCE IMAGING,” filed on June 12, 2023, and to U.S. Provisional Patent Application No. 63/523,655, entitled “SYSTEMS AND METHODS FOR SPINNETWORK LABELED MAGNETIC RESONANCE IMAGING,” filed on June 28, 2023, each of which is incorporated herein by reference in its entirety for all purposes.

TECHNICAL FIELD

[002] The disclosed embodiments generally relate to techniques for use in magnetic resonance imaging (MRI).

BACKGROUND

[003] MRI is a technology with vital applications in chemistry, biology, and medical imaging. Despite these successes, it is recognized that MRI applications may often have limitations on the types of molecules that can be imaged due to the minute nuclear polarization of analytes (typically on the order of 10'⁵). This minute nuclear polarization can result in limited sensitivity in comparison to other analytic techniques such as mass spectrometry. As a consequence, MRI images are typically based on signals acquired from molecules that are in high abundance in a subject, such as water or fat. Thus, it has been difficult to generate MRI images based on signals acquired from less-abundant endogenous or exogenous molecules such as metabolites, drugs, radionuclides, and the like. Since such molecules can be extremely valuable for determining a biological status of a subject, this lack of sensitivity greatly limits the applicability of MRI.

[004] Increasing nuclear spin polarization beyond its thermal equilibrium value can greatly improve magnetic resonance sensitivity. Nuclear spin polarization can be increased using known techniques like parahydrogen induced polarization (PHIP), PHIP-sidearm hydrogenation (PHIP-SAH), PHIP nuclear Overhauser effect system (PHIPNOESYS), signal amplification by reversible exchange (SABRE), and dynamic nuclear polarization (DNP), among others. Using such techniques, the nuclear spin polarization of a material can be drastically increased. For instance, the nuclear spin polarization of a material can sometimes be increased 10,000 times or more. The enhanced nuclear spin polarization can result in a proportional increase in the magnetic resonance signal. As such, hyperpolarization techniques have permitted applications such as determining the rates at which exogenous molecules are metabolized into different metabolic derivatives in a subject. Such information can be indicative of the presence or absence of cancers and other diseases. Thus, hyperpolarization techniques greatly increase the utility of MRI.

[005] Unfortunately, this enhanced polarization decays over time due to the relaxation time of the nuclear spins in the polarized molecules. For many molecules the relaxation time can be on the order of only seconds to minutes. Thus, hyperpolarized molecules will only show dramatic signal enhancements for a short time, after which they revert to their thermal equilibrium polarizations. This makes hyperpolarization techniques much less useful for studies of biological processes that require a long period of time for effects to take hold. For instance, hyperpolarization techniques are ill-suited for studying the biological response of a subject to the administration of a slow-acting drug, radionuclide, or the like.

[006] Another approach for generating MRI images based on signals acquired from low- abundance exogenous molecules is to use deuterium labeling and deuterium MRI. That is, deuterium-labeled exogenous molecules are administered to a subject and a deuterium MRI image is collected. Since endogenous molecules (including water and fat) contain a very small amount of deuterium, the deuterium MRI signal results almost entirely from the administered exogenous molecule and its metabolic derivatives. However, such an approach has serious downsides. First, the nuclear gyromagnetic ratio of deuterium is less than one-sixth that of protons, resulting in a drastic reduction in the MRI signal when compared to the more common proton MRI. This drastically increases the amount of time required to obtain an MRI image. This long period of time can easily exceed a patient’s limits and render the MRI machine unusable for other imaging, making deuterium labeling studies impractical in a clinical setting. Second, the vast majority of MRI machines are built to obtain proton MRI images and lack the equipment required for deuterium imaging. This further limits the applicability of deuterium labeling studies.

[007] Thus, there is a need for systems and methods that allow for the acquisition of MRI images based on relatively low-abundance endogenous or exogenous molecules in a subject or a portion of the subject.

SUMMARY

[008] The systems and methods presented herein allow for the acquisition of MRI images based on relatively low-abundance endogenous or exogenous molecules in a subject or a portion of the subject, such as metabolites, drugs, radionuclides, and the like. The acquired MRI images can then be used to determine a biological status of the subject or the portion of the subject, such as how the subject or the portion of the subject reacts biologically to the administration of the drug or radionuclides, how the subject or the portion of the subject metabolizes the drug or radionuclide, whether the subject or the portion of the subject is necrotic, whether the subject or the portion of the subject is engaging in aerobic or anaerobic metabolism, and the like.

[009] The systems and methods generally identify a target molecule that is either endogenous to the subject or the portion of the subject or that can be administered to (i.e., is exogenous to) the subject or the portion of the subject. When the subject or the portion of the subject is placed in the magnetic field of an MRI machine, all MRI-active molecules in the subject or the portion of the subject generate magnetization. Target magnetization associated with the target molecule or one of its metabolic derivatives is selectively preserved utilizing the J-coupling network of the target molecule or the metabolic derivative. Non-target magnetization associated with other MRI-active molecules is suppressed. When one or more MRI images are later acquired, all or nearly all of the signal results from the target magnetization. Thus, the systems and methods allow for the acquisition of MRI images based on a single relatively low-abundance endogenous or exogenous molecule, free of confounding background signals from higher-abundance molecules such as water. Such clean images may allow for unprecedented insights into biological processes within a subject or a portion of a subject.

[010] The preservation of the target magnetization and the suppression of the non-target magnetization may occur in numerous manners. For instance, the target magnetization may be preserved in a singlet state using certain pulse sequences. While the target magnetization is preserved in the singlet state, it is unaffected by MRI pulse sequences. Thus, MRI pulse sequences such as gradient spoiling or radio-frequency (RF) spoiling can be used to suppress the non-target magnetization. Once the non-target magnetization is suppressed, the target magnetization can be regenerated from the singlet state and an MRI image can be obtained. Since the only remaining magnetization is the target magnetization, the MRI image results from the target molecule or the metabolic derivative of the target molecule.

[Oil] Alternatively, the target magnetization may be preserved in the singlet state, then regenerated into target magnetization having a first phase (e.g., parallel to the magnetic field of the MRI machine). A first MRI image may then be obtained. The target magnetization may again be preserved in the singlet state, then regenerated into target magnetization having a first phase different from the second phase (e.g., anti-parallel to the magnetic field of the MRI machine). A second MRI image may then be obtained. The second MRI image may be subtracted from the first MRI image. Assuming that the same parameters (other than the two different phases of the target magnetization) were used in acquiring the first and second MRI image, subtracting the two images should result in the cancellation of all MRI signals arising due to non-target magnetization. However, since the MRI signals arising from the target magnetization were out of phase, subtracting the two images should result in the preservation of the MRI signal arising from the target magnetization. Thus, the MRI image results from the target molecule or the metabolic derivative of the target molecule.

[012] The methods described above may also be used to obtain MRI images both from a target molecule and one or more of its metabolic derivatives. Such MRI images may be overlaid to determine the relative concentrations of the target molecule and its metabolic derivatives in each voxel of the subject or the portion of the subject. Such information may be obtained over the course of a time series (i.e., by acquiring the MRI images of the target molecule and its metabolic derivatives at multiple points in time) in order to provide dynamic information, such as the rate at which the subject or the portion of the subject is metabolizing a drug or radionuclide, the rates at which specific metabolic pathways are engaged by the subject or the portion of the subject, and so forth.

[013] In some embodiments, the methods described above use target molecules or metabolic derivatives thereof that contain at least two nuclear spins of the same spin species that are coupled by a J-coupling interaction. In other embodiments, the methods described above use target molecules or metabolic derivatives thereof that contain at least two nuclear spins of a different spin species that are coupled by a J-coupling interaction.

[014] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosed embodiments, as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

[015] The accompanying drawings, which comprise a part of this specification, illustrate several embodiments and, together with the description, serve to explain the principles and features of the disclosed embodiments. In the drawings:

[016] FIG. 1 depicts an exemplary method for acquiring MRI images based on relatively low- abundance endogenous or exogenous molecules using two J-coupled nuclear spin of the same type, in accordance with disclosed embodiments.

[017] FIG. 2 depicts an exemplary method for acquiring MRI images by preserving target magnetization and suppressing non-target magnetization via singlet-state preservation using two J-coupled nuclear spins of the same type, in accordance with disclosed embodiments.

[018] FIG. 3 depicts an exemplary method for acquiring MRI images by preserving target magnetization and suppressing non-target magnetization via phase operations using two J- coupled nuclear spins of the same type, in accordance with disclosed embodiments.

[019] FIG. 4 depicts an exemplary method for acquiring MRI images based on multiple relatively low-abundance endogenous or exogenous molecules using two J-coupled nuclear spins of the same type, in accordance with disclosed embodiments.

[020] FIG. 5 depicts an exemplary method for acquiring MRI images based on relatively low- abundance molecules using J-coupled spins of different types, in accordance with disclosed embodiments.

[021] FIG. 6 depicts an exemplary method for acquiring MRI images by preserving target magnetization and suppressing non-target magnetization via magnetization transfer or heteronuclear correlations using J-coupled nuclear spins of different types, in accordance with disclosed embodiments. [022] FIG. 7 depicts an exemplary method for acquiring MRI images by preserving target magnetization and suppressing non-target magnetization via phase operations using J-coupled nuclear spins of different types, in accordance with disclosed embodiments.

[023] FIG. 8 depicts an exemplary method for acquiring MRI images based on multiple relatively low-abundance molecules using J-coupled nuclear spins of different types, in accordance with disclosed embodiments.

[024] FIG. 9A depicts a first exemplary pulse sequence for suppressing magnetization associated with water and protons that are not chemically bonded to carbon- 13 nuclei, in accordance with disclosed embodiments.

[025] FIG. 9B depicts a second exemplary pulse sequence for suppressing magnetization associated with water and protons that are not chemically bonded to carbon- 13 nuclei, in accordance with disclosed embodiments.

[026] FIG. 10 depicts exemplary nuclear magnetic resonance (NMR) spectra of water, fumarate, and malic acid obtained in the absence of and using the pulse sequence depicted in FIG. 9A, in accordance with disclosed embodiments.

[027] FIG. 11 depicts an exemplary time-series graph showing the conversion of fumarate to malic acid, in accordance with disclosed embodiments.

[028] FIG. 12 depicts an exemplary proton NMR spectrum associated with an imaging phantom with and without the pulse sequence depicted in FIG. 9A, in accordance with disclosed embodiments.

DETAILED DESCRIPTION

[029] Reference will now be made in detail to exemplary embodiments, discussed with regards to the accompanying drawings. In some instances, the same reference numbers will be used throughout the drawings and the following description to refer to the same or like parts. Unless otherwise defined, technical and/or scientific terms have the meaning commonly understood by one of ordinary skill in the art. The disclosed embodiments are described in sufficient detail to enable those skilled in the art to practice the disclosed embodiments. It is to be understood that other embodiments may be utilized and that changes may be made without departing from the scope of the disclosed embodiments. Thus, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[030] MRI can be used in a wide variety of applications including, but not limited to, minimally invasive imaging of biological tissues or organisms and even metabolic analyses of biological tissues or organisms. However, MRI applications may often have limitations on the types of molecules that can be imaged due to the minute nuclear polarization of analytes (typically on the order of 1 O'⁵). This minute nuclear polarization can result in limited sensitivity in comparison to other analytic techniques such as mass spectrometry. As a consequence, MRI images are typically based on signals acquired from molecules that are in high abundance in a subject, such as water or fat. Thus, it has been difficult to generate MRI images based on signals acquired from less-abundant endogenous or exogenous molecules such as metabolites, drugs, radionuclides, and the like. Since such molecules can be extremely valuable for determining a biological status of a subject, this lack of sensitivity greatly limits the applicability of MRI.

[031] The disclosed embodiments allow for the acquisition of MRI images based on relatively low-abundance endogenous or exogenous molecules in a subject or a portion of the subject, such as metabolites, drugs, radionuclides, and the like. The acquired MRI images can then be used to determine a biological status of the subject or the portion of the subject, such as how the subject or the portion of the subject reacts biologically to the administration of the drug or radionuclides, how the subj ect or the portion of the subj ect metabolizes the drug or radionuclide, whether the subject or the portion of the subject is necrotic, whether the subject or the portion of the subject is engaging in aerobic or anaerobic metabolism, and the like. [032] The disclosed embodiments generally identify a target molecule that is either endogenous to the subject or the portion of the subject or that can be administered to (i.e., is exogenous to) the subject or the portion of the subject. When the subject or the portion of the subject is placed in the magnetic field of an MRI machine, all MRI-active molecules in the subject or the portion of the subject generate magnetization. Target magnetization associated with the target molecule or one of its metabolic derivatives is selectively preserved utilizing the J-coupling network of the target molecule or the metabolic derivative. Non-target magnetization associated with other MRI-active molecules is suppressed. When one or more MRI images are later acquired, all or nearly all of the signal results from the target magnetization. Thus, the systems and methods allow for the acquisition of MRI images based on a single relatively low- abundance endogenous or exogenous molecule, free of confounding background signals from higher-abundance molecules such as water. Such clean images may allow for unprecedented insights into biological processes within a subject or a portion of a subject.

[033] In some embodiments, the methods described above use target molecules or metabolic derivatives thereof that contain at least two proton spins coupled by a J-coupling interaction. In other embodiments, the methods described above use target molecules or metabolic derivatives thereof that contain at least a proton spin and a carbon- 13 spin coupled by a J-coupling interaction.

Definitions

[034] As used in the present disclosure, the phrase “or” refers to both conjunctive and disjunctive meanings, unless such a definition is impossible in a given context. For instance, the phrase “A or B” refers to A alone, B alone, or A and B, unless any such meaning is impossible. Similarly, the phrase “A, B, or C” refers to A alone, B alone, C alone, A and B but not C, A and C but not B, B and C but not A, or A, B, and C, unless any such meaning is impossible. [035] As used in the present disclosure, the phrase “subject” refers to any human, non-human primate, horse, pig, dog, cat, rat, mouse, or other animal that is subjected to an MRI imaging procedure.

[036] As used in the present disclosure, the phrase “portion of a/the subject” refers to any portion of a subject’s body, including but not limited to a head, brain, chest, heart, lung, torso, stomach, intestine, arm, hand, pelvis, hip, buttocks, leg, foot, or any portion thereof.

[037] As used in the present disclosure, the phrase “target molecule” refers to any molecule from which an MRI image can be obtained using the systems and methods described herein, or to any molecule having a metabolic derivative from which an MRI image can be obtained using the systems and methods described herein. Target molecules include, but are not limited to, biologically active molecules, antibodies, drugs, antibody-drug conjugates, radionuclides, radionuclide-drug conjugates, fumarate, glucose, and any carbon-13 labeled, nitrogen-15 labeled, oxygen- 17 labeled, partially deuterated, or fully deuterated analog thereof. Target molecules may be endogenous or exogenous to a subject or a portion of a subject.

[038] As used in the present disclosure, the phrase “metabolic derivative” refers to any molecule that is formed with a subject or a portion of a subject by metabolism of a target molecule. Metabolic derivatives include, but are not limited to, metabolic derivatives of a biologically active molecule, metabolic derivatives of a drug, metabolic derivatives of an antibody-drug conjugate, metabolic derivative of a radionuclide-drug conjugates, malate, glutamate, and any carbon- 13 labeled, nitrogen- 15 labeled, oxygen- 17 labeled, partially deuterated, or fully deuterated analog thereof.

[039] As used in the present disclosure, the phrase “endogenous” refers to any molecule that naturally occurs within a subject or a portion of a subject.

[040] As used in the present disclosure, the phrase “exogenous” refers to any molecule that does not naturally occur within a subject or a portion of a subject. [041] As used in the present disclosure, the phrase “MRI-active molecules” refers to any molecules that give rise to an MRI signal. MRI-active molecules generally comprise at least one nucleus that is under study in an MRI procedure. For instance, in proton MRI imaging, MRI-active molecules comprise any molecules that contain at least one proton.

[042] As used in the present disclosure, the phrase “target magnetization” refers to magnetization associated with a target molecule or a metabolic derivative of a target molecule from which an MRI image is to be obtained.

[043] As used in the present disclosure, the phrase “derivative target magnetization” refers to magnetization associated with a metabolic derivative of a target molecule from which an MRI image is to be obtained.

[044] As used in the present disclosure, the phrase “non-target magnetization” refers to magnetization associated with MRI-active molecules other than the molecule from which an MRI image is to be obtained. In some embodiments, the non-target magnetization is associated with molecules in the subject or the portion of the subject that would contribute a relatively strong or confounding MRI signal unless suppressed using the systems and methods described herein. For instance, in some embodiments, the non-target magnetization is associated with water or fat in the subject or the portion of the subject.

[045] As used in the present disclosure, the phrase “carbon- 13 labeled” refers to any molecule containing at least one carbon-13 atom where a carbon-12 atom would otherwise be expected due to the high natural abundance of carbon-12. A carbon-13 labeled molecule generally contains a higher fraction of carbon- 13 atoms at a particular chemical site than would be expected were the molecule to incorporate carbon- 13 at natural abundance (approximately 1.1%). Thus, a carbon-13 labeled molecule may comprise at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more carbon-13 atoms at a particular chemical site, and at most 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or less carbon-12 atoms at that particular chemical site.

[046] As used in the present disclosure, the phrase “isotopically enriched carbon- 13 nuclear spin” refers to any carbon- 13 nuclear spin in a carbon- 13 labeled molecule.

[047] As used in the present disclosure, the phrase “nitrogen- 15 labeled” refers to any molecule containing at least one nitrogen-15 atom where a nitrogen-14 atom would otherwise be expected due to the high natural abundance of nitrogen-14. A nitrogen-15 labeled molecule generally contains a higher fraction of nitrogen- 15 atoms at a particular chemical site than would be expected were the molecule to incorporate nitrogen- 15 at natural abundance (approximately 0.35%). Thus, a nitrogen- 15 labeled molecule may comprise at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more nitrogen-15 atoms at a particular chemical site, and at most 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or less nitrogen- 14 atoms at that particular chemical site.

[048] As used in the present disclosure, the phrase “isotopically enriched nitrogen- 15 nuclear spin” refers to any nitrogen- 15 nuclear spin in a nitrogen- 15 labeled molecule.

[049] As used in the present disclosure, the phrase “oxygen-17 labeled” refers to any molecule containing at least one oxygen- 17 atom where an oxygen- 16 atom would otherwise be expected due to the high natural abundance of oxygen- 16. An oxygen- 17 labeled molecule generally contains a higher fraction of oxygen- 17 atoms at a particular chemical site than would be expected were the molecule to incorporate oxygen- 17 at natural abundance (approximately 0.04%). Thus, an oxygen-17 labeled molecule may comprise at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more oxygen- 17 atoms at a particular chemical site, and at most 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or less oxygen-17 atoms at that particular chemical site.

[050] As used in the present disclosure, the phrase “isotopically enriched oxygen- 17 nuclear spin” refers to any oxygen- 17 nuclear spin in an oxygen- 17 labeled molecule.

[051] As used in the present disclosure, the phrase “partially deuterated” refers to any molecule containing at least one deuterium atom where a proton would otherwise be expected due to the high natural abundance of protons.

[052] As used in the present disclosure, the phrase “fully deuterated” refers to any molecule containing a deuterium atom at every site where a proton would otherwise be expected due to the high natural abundance of protons.

[053] As used in the present disclosure, the phrase “biological status” refers to any trait or attribute associated with a subject or a portion of a subject that offers insight into biological processes occurring within the subject or the portion of the subject. Biological statuses include, but are not limited to, a biological reaction of the subject or the portion of the subject to a biologically active molecule or a metabolic derivative of a biologically active molecule, a biological reaction of the subject or the portion of the subject to an antibody, a biological reaction of the subject or the portion of the subject to a drug or a metabolic derivative of a drug, a biological reaction of the subject or the portion of the subject to an antibody-drug conjugate or a metabolic derivative of an antibody-drug conjugate, a biological reaction of the subject or the portion of the subject to a radionuclide, a biological reaction of the subject or the portion of the subject to a radionuclide-drug conjugate or a metabolic derivative of a radionuclide-drug conjugate, a presence or absence of necrosis in the subject or the portion of the subject, an aerobic status of the subject or the portion of the subject, and an anaerobic status of the subject or the portion of the subject.

Methods for acquiring MRI images based on relatively low-abundance endogenous or exogenous molecules using two J-coupled nuclear spins of the same type [054] FIG. 1 depicts an exemplary method 100 for acquiring MRI images based on relatively low-abundance endogenous or exogenous molecules using two J-coupled nuclear spins of the same type. At 110, a target molecule is identified in a subject or a portion of a subject. In some embodiments, the target molecule comprises a first nuclear spin and a second nuclear spin. In some embodiments, the first nuclear spin and the second nuclear spin are of the same spin species. That is, in some embodiments, the first nuclear spin and the second nuclear spin are the same nuclear isotope. In some embodiments, the first nuclear spin and the second nuclear spin each comprise a spin- 1/2 nuclear spin. In some embodiments, the first nuclear spin and the second nuclear spin each comprise a proton nuclear spin. In some embodiments, the first nuclear spin and the second nuclear spin each comprise a carbon- 13 nuclear spin. In some embodiments, the first nuclear spin and the second nuclear spin each comprise an isotopically enriched carbon- 13 nuclear spin. In some embodiments, the first nuclear spin and the second nuclear spin each comprise a nitrogen- 15 nuclear spin. In some embodiments, the first nuclear spin and the second nuclear spin each comprise an isotopically enriched nitrogen- 15 nuclear spin. In some embodiments, the first nuclear spin and the second nuclear spin couple to form a spin-1 system comprising a singlet state and a triplet state.

[055] In some embodiments, the first nuclear spin and the second nuclear spin are coupled by an electron-mediated J-coupling. In some embodiments, the J-coupling between the first nuclear spin and the second nuclear spin is at least about 0.1 hertz (Hz), 0.2 Hz, 0.3 Hz, 0.4 Hz, 0.5 Hz, 0.6 Hz, 0.7 Hz, 0.8 Hz, 0.9 Hz, 1 Hz, 2 Hz, 3 Hz, 4 Hz, 5 Hz, 6 Hz, 7 Hz, 8 Hz, 9 Hz, 10 Hz, 20 Hz, 30 Hz, 40 Hz, 50 Hz, 60 Hz, 70 Hz, 80 Hz, 90 Hz, 100 Hz, 110 Hz, 120 Hz, 130 Hz, 140 Hz, 150 Hz, 160 Hz, 170 Hz, 180 Hz, 190 Hz, 200 Hz, or more, at most about 200 Hz, 190 Hz, 180 Hz, 170 Hz, 160 Hz, 150 Hz, 140 Hz, 130 Hz, 120 Hz, 110 Hz, 100 Hz, 90 Hz, 80 Hz, 70 Hz, 60 Hz, 50 Hz, 40 Hz, 30 Hz, 20 Hz, 10 Hz, 9 Hz, 8 Hz, 7 Hz, 6 Hz, 5 Hz, 4 Hz, 3

Hz, 2 Hz, 1 Hz, 0.9 Hz, 0.8 Hz, 0.7 Hz, 0.6 Hz, 0.5 Hz, 0.4 Hz, 0.3 Hz, 0.2 Hz, 0.1 Hz, or less, or within a range defined by any two of the preceding values. In some embodiments, the first nuclear and the second nuclear are covalently connected to the same carbon atom, e.g. in CH2 groups. In some embodiments, the first nuclear and the second nuclear are covalently attached to neighboring carbon atoms.

[056] In some embodiments, target magnetization associated with the target molecule (or a metabolic derivative of the target molecule) is selectively preserved (e.g., in a singlet state) by utilizing the J-coupling network of the target molecule (or the metabolic derivative of the target molecule). In some embodiments, such selective preservation requires a breaking of the symmetry between the first nuclear spin and the second nuclear spin in order to selectively manipulate the spin states of the first nuclear spin and the second nuclear spin (e.g., by coupling between the singlet state and triplet states). Thus, in some embodiments, the target molecule (or the metabolic derivative of the target molecule) either displays a chemical shift difference between the first nuclear spin and the second nuclear spin or comprises a third nuclear spin that couples differently to the first nuclear spin and the second nuclear spin.

[057] Thus, in some embodiments, the target molecule (or the metabolic derivative of the target molecule) displays a chemical shift difference between the first nuclear spin and the second nuclear spin. In some embodiments, the chemical shift difference breaks a symmetry between the first nuclear spin and the second nuclear spin and allows selective preservation of the target magnetization, for example by coupling between the singlet state and triplet states of the first and second nuclear spins. In some embodiments, the chemical shift difference is at least about 0.001 parts per million (ppm), 0.002 ppm, 0.003 ppm, 0.004 ppm, 0.005 ppm, 0.006 ppm, 0.007 ppm, 0.008 ppm, 0.009 ppm, 0.01 ppm, 0.02 ppm, 0.03 ppm, 0.04 ppm, 0.05 ppm, 0.06 ppm, 0.07 ppm, 0.08 ppm, 0.09 ppm, 0.1 ppm, 0.2 ppm, 0.3 ppm, 0.4 ppm, 0.5 ppm, 0.6 ppm, 0.7 ppm, 0.8 ppm, 0.9 ppm, 1 ppm, 2 ppm, 3 ppm, 4 ppm, 5 ppm, 6 ppm, 7 ppm, 8 ppm, 9 ppm, 10 ppm, or more, at most about 10 ppm, 9 ppm, 8 ppm, 7 ppm, 6 ppm, 5 ppm, 4 ppm, 3 ppm, 2 ppm, 1 ppm, 0.9 ppm, 0.8 ppm, 0.7 ppm, 0.6 ppm, 0.5 ppm, 0.4 ppm, 0.3 ppm, 0.2 ppm, 0.1 ppm, 0.09 ppm, 0.08 ppm, 0.07 ppm, 0.06 ppm, 0.05 ppm, 0.04 ppm, 0.03 ppm, 0.02 ppm, 0.01 ppm, 0.009 ppm, 0.008 ppm, 0.007 ppm, 0.006 ppm, 0.005 ppm, 0.004 ppm, 0.003 ppm, 0.002 ppm, 0.001 ppm, or less, or within a range defined by any two of the preceding values.

[058] Alternatively or in combination, in some embodiments, the target molecule (or the metabolic derivative of the target molecule) comprises a third nuclear spin. In some embodiments, the third nuclear spin comprises a proton nuclear spin, a deuterium nuclear spin, a carbon- 13 nuclear spin, a nitrogen- 14 nuclear spin, a nitrogen- 15 nuclear spin, an oxygen- 17 nuclear spin, a fluorine-19 nuclear spin, a phosphorous-31 nuclear spin, a sulfur-33 nuclear spin, a chlorine-35 nuclear spin, a chlorine-37 nuclear spin, or the like. In some embodiments, the third nuclear spin couples to the first nuclear spin and the second nuclear spin via J-coupling interactions. In some embodiments, the J-coupling between the third nuclear spin and the first nuclear spin is different than the J-coupling between the third nuclear spin and the second nuclear spin. In some embodiments, the difference in J-couplings breaks a symmetry between the first nuclear spin and the second nuclear spin and allows selective preservation of the target magnetization, as described herein.

[059] In some embodiments, the target molecule is endogenous to the subject or the portion of the subject. Thus, in some embodiments, the target molecule need not be administered to the subject or the portion of the subject. In other embodiments, the target molecule is exogenous to the subject or the portion of the subject. Thus, in some embodiments, the target molecule is administered to the subject or the portion of the subject.

[060] At 120, the subject or the portion of the subject is subjected to a magnetic field in an MRI machine. In some embodiments, the magnetic field has an average magnitude (within an active area of the magnetic field, such as within the bore of the MRI machine) of at least about 0.1 tesla (T), 0.2 T, 0.3 T, 0.4 T, 0.5 T, 0.6 T, 0.7 T, 0.8 T, 0.9 T, 1 T, 1.25 T, 1.5 T, 1.75 T, 2 T, 2.5 T, 3 T, 3.5 T, 4 T, 4.5 T, 5 T, 5.5 T, 6 T, 6.5 T, 7 T, 7.5 T, 8 T, 8.5 T, 9 T, 9.5 T, 10 T,

11 T, 12 T, 13 T, 14 T, 15 T, 16 T, 17 T, 18 T, 19 T, 20 T, or more, at most about 20 T, 19 T,

18 T, 17 T, 16 T, 15 T, 14 T, 13 T, 12 T, 11 T, 10 T, 9.5 T, 9 T, 8.5 T, 8 T, 7.5 T, 7 T, 6.5 T, 6

T, 5.5 T, 5 T, 4.5 T, 4 T, 3.5 T, 3 T, 2.5 T, 2 T, 1.75 T, 1.5 T, 1.25 T, 1 T, 0.9 T, 0.8 T, 0.7 T,

0.6 T, 0.5 T, 0.4 T, 0.3 T, 0.2 T, 0.1 T, or less, or an average magnitude that is within a range defined by any two of the preceding values. In some embodiments, subjecting the subject or the portion of the subject to the magnetic field generates target magnetization in the target molecule or the metabolic derivative of the target molecule and generates non-target magnetization in other MRI-active molecules in the subject or the portion of the subject.

[061] In some embodiments, step 120 is performed a predetermined time period after step 110. In some embodiments, performing step 120 a predetermined time period after step 110 permits the subject or the portion of the subject to display a biological response to the target molecule. That is, in some embodiments, during the predetermined time period, the target molecule is permitted to obtain a biological response in the subject or the portion of the subject. In some embodiments, the biological response is indicative of a biological status of the subject or the portion of the subject. In some embodiments, the predetermined time period is at least about 1 minute (min), 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 1 hour (h), 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, or more, at most about 12 h, 11 h, 10 h, 9 h, 8 h, 7 h, 6 h, 5 h, 4 h, 3 h, 2 h, 1 h, 55 min, 50 min, 45 min, 40 min, 35 min, 30 min, 25 min, 20 min, 15 min, 10 min, 9 min, 8 min, 7 min, 6 min, 5 min, 4 min, 3 min, 2 min, 1 min, or less, or within a range defined by any two of the preceding values.

[062] At 130, one or more pulse sequences are performed to preserve the target magnetization and to suppress the non-target magnetization. In some embodiments, the one or more pulse sequences utilize a J-coupling network of the target molecule (or the metabolic derivative of the target molecule) to selectively preserve the target magnetization. In some embodiments, the one or more pulse sequences are configured to reduce the non-target magnetization by a factor of at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000,

6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, or more, a factor of at most about 100,000, 90,000, 80,000, 70,000, 60,000, 50,000, 40,000, 30,000, 20,000, 10,000, 9,000, 8,000, 7,000, 6,000, 5,000, 4,000, 3,000, 2,000, 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 100, or less, or a factor that is within a range defined by any two of the preceding values. In some embodiments, the reduction in the nontarget magnetization is in comparison to the non-target magnetization that would be expected in the absence of the pulse sequences described herein. In some embodiments, the one or more pulses sequences are configured to suppress the non-target magnetization such that most or substantially all of the remaining magnetization comprises target magnetization. In some embodiments, the one or more pulse sequences correspond to the one or more pulse sequences described herein with respect to step 210 of FIG. 2. In some embodiments, the one or more pulse sequences correspond to the one or more first pulse sequences and the one or more second pulse sequences described herein with respect to steps 310 and 330, respectively, of FIG. 3.

[063] At 140, one or more MRI images of the target molecule or the metabolic derivative of the target molecule in the subject or the portion of the subject are obtained. In some embodiments, the one or more MRI images correspond to the one or more MRI images described herein with respect to step 220 of FIG. 2. In some embodiments, the one or more MRI images correspond to the one or more first MRI images, the one or more second MRI images, and the one or more background- subtracted MRI images described herein with respect to steps 320, 340, and 350, respectively, of FIG. 3.

[064] At 150, a biological status of the subj ect or the portion of the subj ect is determined based on the one or more MRI images. Methods for acquiring MRI images by preserving target magnetization and suppressing non-target magnetization via singlet-state preservation using two J-coupled nuclear spins of the same type

[065] FIG. 2 depicts an exemplary method 200 for acquiring MRI images by preserving target magnetization and suppressing non-target magnetization via singlet-state preservation using two J-coupled nuclear spins of the same type. At 210, one or more pulse sequences are performed to preserve the target magnetization in a singlet state, suppress the non-target magnetization, and regenerate the target magnetization from the singlet state. In some embodiments, the one or more pulse sequences utilize the J-coupling network of the target molecule or the metabolic derivative of the target molecule. In some embodiments, the one or more pulse sequences comprise one or more PulsePol pulse sequences (as described in M. Korzeczek et al, “Towards a unified picture of polarization transfer - equivalence of DNP and PHIP,” arXiv:2303.07478 (2023), which is incorporated herein by reference in its entirety for all purposes), one or more gradient spoiling sequences (as described in B. Hargreaves, “Rapid gradient-echo imaging,” J. Mag. Res. 36(6), 1300-1313 (2012), which is incorporated herein by reference in its entirety for all purposes), one or more radio-frequency (RF) spoiling pulse sequences (as described in B. Hargreaves, “Rapid gradient-echo imaging,” J. Mag. Res. 36(6), 1300-1313 (2012), which is incorporated herein by reference in its entirety for all purposes), or any combination thereof. In some embodiments, preserving the target magnetization in the singlet state prevents the target magnetization from being affected by spin operations (such as gradient spoiling or RF spoiling) that affects the non-preserved, non-target magnetization. Then, the non-target magnetization can be suppressed using spin operations such as the gradient spoiling or RF spoiling. Finally, the target magnetization can be regenerated from the singlet state. Thus, following step 210, most or substantially all of the remaining magnetization is target magnetization. [066] At 220, one or more MRI images of the target molecule or the metabolic derivative of the target molecule in the subject or the portion of the subject are obtained.

Methods for acquiring MRI images by preserving target magnetization and suppressing non-target magnetization via phase operations using two J-coupled nuclear spins of the same type

[067] FIG. 3 depicts an exemplary method 300 for acquiring MRI images by preserving target magnetization and suppressing non-target magnetization via phase operations using two J- coupled nuclear spins of the same type. At 310, one or more first pulse sequences are performed to preserve the target magnetization in a singlet state and to generate first target magnetization having a first phase from the singlet state. In some embodiments, the first phase is selected such that the first magnetization is aligned parallel to the magnetic field. In some embodiments, the one or more first pulse sequences utilize the J-coupling network of the target molecule or the metabolic derivative of the target molecule. In some embodiments, the one or more first pulse sequences comprise one or more PulsePol pulse sequences, one or more gradient spoiling sequences, one or more radio-frequency (RF) spoiling pulse sequences, or any combination thereof.

[068] At 320, one or more first MRI images of the target molecule or the metabolic derivative of the target molecule in the subject or the portion of the subject are obtained based on the first target magnetization.

[069] At 330, one or more second pulse sequences are performed to preserve the target magnetization in a singlet state and to generate second target magnetization having a second phase from the singlet state. In some embodiments, the second phase is different from the first phase. In some embodiments, the second phase is selected such that the second magnetization is aligned anti-parallel to the magnetic field. In some embodiments, the one or more second pulse sequences utilize the J-coupling network of the target molecule or the metabolic derivative of the target molecule. In some embodiments, the one or more second pulse sequences comprise one or more PulsePol pulse sequences, one or more gradient spoiling sequences, one or more radio-frequency (RF) spoiling pulse sequences, or any combination thereof.

[070] At 340, one or more second MRI images of the target molecule or the metabolic derivative of the target molecule in the subject or the portion of the subject are obtained based on the second target magnetization.

[071] At 350, the one or more second MRI images are subtracted from the one or more first MRI images to obtain one or more background-subtracted MRI images. In some embodiments, the one or more background-subtracted MRI images comprise suppressed MRI signals from the non-target magnetization. That is, assuming that the first and second pulse sequences are substantially identical (with the exception of the first and second phases imparted to the target magnetization), the MRI signals resulting from the non-target magnetization will be substantially identical in the first and second MRI images. However, the MRI signals resulting from the target magnetization will be out of phase (e.g., 180 degrees out of phase when the first and second target magnetizations are aligned parallel and anti-parallel to the magnetic field, respectively). Thus, when the one or more second MRI images are subtracted from the one or more first MRI images, the MRI signals resulting from the non-target magnetization will substantially cancel out. On the other hand, the MRI signals resulting from the target magnetization will remain. For instance, when the first and second target magnetizations are aligned parallel and anti-parallel to the magnetic field, the remaining MRI signal will be approximately twice as strong as the MRI signals resulting from the target magnetization in the one or more first MRI images or the one or more second MRI images.

[072] Although FIG. 3 describes method 300 as utilizing first target magnetization that is aligned parallel to the magnetic field and second target magnetization that is aligned anti- parallel to the magnetic field, the disclosure is not intended to be so limiting. The first and second phases may have any possible values, so long as they are different. For instance, the first phase may be chosen such that the first target magnetization is aligned anti-parallel to the magnetic field and the second target magnetization is aligned parallel to the magnetic field. The second phase may be different from the first phase by at least about 10 degrees, 20 degrees, 30 degrees, 40 degrees, 50 degrees, 60 degrees, 70 degrees, 80 degrees, 90 degrees, 100 degrees, 110 degrees, 120 degrees, 130 degrees, 140 degrees, 150 degrees, 160 degrees, 170 degrees, 180 degrees, or more, at most about 180 degrees, 170 degrees, 160 degrees, 150 degrees, 140 degrees, 130 degrees, 120 degrees, 110 degrees, 100 degrees, 90 degrees, 80 degrees, 70 degrees, 60 degrees, 50 degrees, 40 degrees, 30 degrees, 20 degrees, 10 degrees, or less, or by an angle that is within a range defined by any two of the preceding values.

Methods for acquiring MRI images based on multiple relatively low-abundance endogenous or exogenous molecules using two J-coupled nuclear spins of the same type

[073] FIG. 4 depicts an exemplary method 400 for acquiring MRI images based on multiple relatively low-abundance endogenous or exogenous molecules using two J-coupled nuclear spins of the same type. At 410, a target molecule is identified in a subject or a portion of a subject. In some embodiments, the target molecule comprises a first nuclear spin and a second nuclear spin. In some embodiments, the first nuclear spin and the second nuclear spin are coupled by an electron-mediated J-coupling. In some embodiments, the J-coupling between the first nuclear spin and the second nuclear spin is any J-coupling described herein with respect to step 110 of FIG. 1.

[074] In some embodiments, target magnetization associated with the target molecule is selectively preserved (e.g., in a singlet state) by utilizing the J-coupling network of the target molecule. In some embodiments, such selective preservation requires a breaking of the symmetry between the first nuclear spin and the second nuclear spin, as described herein. Thus, in some embodiments, the target molecule either displays a chemical shift difference between the first nuclear spin and the second nuclear spin or comprises a third nuclear spin that couples differently to the first nuclear spin and the second nuclear spin.

[075] Thus, in some embodiments, the target molecule displays a chemical shift difference between the first nuclear spin and the second nuclear spin. In some embodiments, the chemical shift difference breaks a symmetry between the first nuclear spin and the second nuclear spin and allows selective preservation of the target magnetization. In some embodiments, the chemical shift difference is any chemical shift difference described herein with respect to step 110 of FIG. 1.

[076] Alternatively or in combination, in some embodiments, the target molecule comprises a third nuclear spin. In some embodiments, the third nuclear spin comprises a third proton nuclear spin, a deuterium nuclear spin, a carbon- 13 nuclear spin, a nitrogen- 14 nuclear spin, a nitrogen- 15 nuclear spin, an oxygen- 17 nuclear spin, a fluorine- 19 nuclear spin, a phosphorous- 31 nuclear spin, a sulfur-33 nuclear spin, a chlorine-35 nuclear spin, a chlorine-37 nuclear spin, or the like. In some embodiments, the third nuclear spin couples to the first nuclear spin and the second nuclear spin via J-coupling interactions. In some embodiments, the J-coupling between the third nuclear spin and the first nuclear spin is different than the J-coupling between the third nuclear spin and the second nuclear spin. In some embodiments, the difference in J-couplings breaks a symmetry between the first nuclear spin and the second nuclear spin and allows selective preservation of the target magnetization.

[077] In some embodiments, the target molecule is endogenous to the subject or the portion of the subject. Thus, in some embodiments, the target molecule need not be administered to the subject or the portion of the subject. In other embodiments, the target molecule is exogenous to the subject or the portion of the subject. Thus, in some embodiments, the target molecule is administered to the subject or the portion of the subject. [078] At 420, at least a portion of the target molecule is permitted to be converted to a derivative molecule (e.g., any metabolic derivative of the target molecule described herein). In some embodiments, the derivative molecule comprises a first nuclear spin and a second nuclear spin. In some embodiments, the first nuclear spin and the second nuclear spin are coupled by an electron-mediated J-coupling. In some embodiments, the J-coupling between the first nuclear spin and the second nuclear spin is any J-coupling described herein with respect to step 110 of FIG. 1.

[079] In some embodiments, derivative target magnetization associated with the derivative molecule is selectively preserved (e.g., in a singlet state) by utilizing the J-coupling network of the derivative molecule. In some embodiments, such selective preservation requires a breaking of the symmetry between the first nuclear spin and the second nuclear spin, as described herein. Thus, in some embodiments, the derivative molecule either displays a chemical shift difference between the first nuclear spin and the second nuclear spin or comprises a third nuclear spin that couples differently to the first nuclear spin and the second nuclear spin.

[080] Thus, in some embodiments, the derivative molecule displays a chemical shift difference between the first nuclear spin and the second nuclear spin. In some embodiments, the chemical shift difference breaks a symmetry between the first nuclear spin and the second nuclear spin and allows selective preservation of the target magnetization. In some embodiments, the chemical shift difference is any chemical shift difference described herein with respect to step 110 of FIG. 1.

[081] Alternatively or in combination, in some embodiments, the derivative molecule comprises a third nuclear spin. In some embodiments, the third nuclear spin comprises any third nuclear spin described herein with respect to step 110 of FIG. 1. In some embodiments, the third nuclear spin couples to the first nuclear spin and the second nuclear spin via J-coupling interactions. In some embodiments, the J-coupling between the third nuclear spin and the first nuclear spin is different than the J-coupling between the third nuclear spin and the second nuclear spin. In some embodiments, the difference in J-couplings breaks a symmetry between the first nuclear spin and the second nuclear spin and allows selective preservation of the target magnetization.

[082] In some embodiments, the derivative molecule is endogenous to the subject or the portion of the subject. In other embodiments, the derivative molecule is exogenous to the subject or the portion of the subject.

[083] At 430, the subject or the portion of the subject is subjected to a magnetic field in an MRI machine. In some embodiments, the magnetic field has any average magnitude described herein with respect to step 120 of FIG. 1. In some embodiments, subjecting the subject or the portion of the subject to the magnetic field generates target magnetization in the target molecule, generates derivative target magnetization in the derivative molecule, and generates non-target magnetization in other MRI-active molecules in the subject or the portion of the subject.

[084] In some embodiments, step 430 is performed a predetermined time period after step 410. In some embodiments, performing step 430 a predetermined time period after step 410 permits the subject or the portion of the subject to display a biological response to the target molecule. That is, in some embodiments, during the predetermined time period, the target molecule is permitted to obtain a biological response in the subject or the portion of the subject. In some embodiments, the biological response is indicative of a biological status of the subject or the portion of the subject. In some embodiments, the predetermined time period comprises any predetermined time period described herein with respect to step 120 of FIG. 1.

[085] At 440, one or more pulse sequences are performed to preserve the target magnetization and to suppress the non-target magnetization and the derivative target magnetization. In some embodiments, the one or more pulse sequences utilize a J-coupling network of the target molecule to selectively preserve the target magnetization. In some embodiments, the one or more pulse sequences are configured to suppress any amount of non-target magnetization and derivative target magnetization described herein with respect to step 130 of FIG. 1. In some embodiments, the one or more pulses sequences are configured to suppress the non-target magnetization and the derivative target magnetization such that most or substantially all of the remaining magnetization comprises target magnetization. In some embodiments, the one or more pulse sequences correspond to the one or more pulse sequences described herein with respect to step 210 of FIG. 2. In some embodiments, the one or more pulse sequences correspond to the one or more first pulse sequences and the one or more second pulse sequences described herein with respect to steps 310 and 330, respectively, of FIG. 3.

[086] At 450, one or more MRI images of the target molecule in the subject or the portion of the subject are obtained. In some embodiments, the one or more MRI images correspond to the one or more MRI images described herein with respect to step 220 of FIG. 2. In some embodiments, the one or more MRI images correspond to the one or more first MRI images, the one or more second MRI images, and the one or more background- subtracted MRI images described herein with respect to steps 320, 340, and 350, respectively, of FIG. 3.

[087] At 460, one or more pulse sequences are performed to preserve the derivative target magnetization and to suppress the non-target magnetization and the target magnetization. In some embodiments, the one or more pulse sequences utilize a J-coupling network of the derivative molecule to selectively preserve the derivative target magnetization. In some embodiments, the one or more pulse sequences are configured to suppress any amount of non- target magnetization and target magnetization described herein with respect to step 130 of FIG.

1. In some embodiments, the one or more pulses sequences are configured to suppress the non- target magnetization and the target magnetization such that most or substantially all of the remaining magnetization comprises derivative target magnetization. In some embodiments, the one or more pulse sequences correspond to the one or more pulse sequences described herein with respect to step 210 of FIG. 2. In some embodiments, the one or more pulse sequences correspond to the one or more first pulse sequences and the one or more second pulse sequences described herein with respect to steps 310 and 330, respectively, of FIG. 3.

[088] At 470, one or more MRI images of the derivative molecule in the subject or the portion of the subject are obtained. In some embodiments, the one or more MRI images correspond to the one or more MRI images described herein with respect to step 220 of FIG. 2. In some embodiments, the one or more MRI images correspond to the one or more first MRI images, the one or more second MRI images, and the one or more background- subtracted MRI images described herein with respect to steps 320, 340, and 350, respectively, of FIG. 3.

[089] At 480, a biological status of the subj ect or the portion of the subj ect is determined based on the one or more MRI images of the target molecule and the one or more MRI images of the derivative molecule.

Methods for acquiring MRI images based on relatively low-abundance molecules using J- coupled nuclear spins of different types

[090] FIG. 5 depicts an exemplary method 500 for acquiring MRI images based on relatively low-abundance molecules using J-coupled spins of different types. At 510, a target molecule is provided (e.g., administered, injected, digested, or otherwise introduced) in a subject or a portion of a subject. In some embodiments, the target molecule comprises a first nuclear spin and a second nuclear spin. In some embodiments, the second nuclear spin is of a different spin species from the first nuclear spin. That is, in some embodiments, the first nuclear spin and the second nuclear spin are different nuclear isotopes. For instance, in some embodiments, the first nuclear spin comprises a proton nuclear spin and the second nuclear spin comprises a carbon- 13 nuclear spin, an isotopically enriched carbon-13 nuclear spin, a nitrogen-14 nuclear spin, a nitrogen-15 nuclear spin, an isotopically enriched nitrogen-15 nuclear spin, an oxygen-17 nuclear spin, an isotopically enriched oxygen- 17 nuclear spin, a fluorine- 19 nuclear spin, or a phosphorus-31 nuclear spin. As another example, in some embodiments, the first nuclear spin comprises a carbon- 13 nuclear spin or an isotopically enriched carbon- 13 nuclear spin and the second nuclear spin comprises a proton nuclear spin, a nitrogen-14 nuclear spin, a nitrogen-15 nuclear spin, an isotopically enriched nitrogen- 15 nuclear spin, an oxygen- 17 nuclear spin, an isotopically enriched oxygen-17 nuclear spin, a fluorine-19 nuclear spin, or a phosphorus-31 nuclear spin. Thus, in some embodiments, the target molecule is carbon-13 labeled, nitrogen- 15 labeled, or oxygen- 17 labeled.

[091] In some embodiments, the first nuclear spin and the second nuclear spin are coupled by an electron-mediated J-coupling. In some embodiments, the J-coupling between the first nuclear spin and the second nuclear spin is at least 10 Hz, 20 Hz, 30 Hz, 40 Hz, 50 Hz, 60 Hz, 70 Hz, 80 Hz, 90 Hz, 100 Hz, 110 Hz, 120 Hz, 130 Hz, 140 Hz, 150 Hz, 160 Hz, 170 Hz, 180 Hz, 190 Hz, 200 Hz, or more, at most about 200 Hz, 190 Hz, 180 Hz, 170 Hz, 160 Hz, 150 Hz, 140 Hz, 130 Hz, 120 Hz, 110 Hz, 100 Hz, 90 Hz, 80 Hz, 70 Hz, 60 Hz, 50 Hz, 40 Hz, 30 Hz, 20 Hz, 10 Hz, or less, or within a range defined by any two of the preceding values. In some embodiments, the first nuclear spin is chemically bonded (e.g., covalently bonded) to the second nuclear spin (i.e., the first nuclear spin and the second nuclear spin are located one chemical bond away from one another). For instance, in some embodiments, the first nuclear spin and the second nuclear spin form constituents of a C-H bond, N-H bond, O-H bond, P-H bond, C- N bond, C-0 bond, C-F bond, C-P bond, or the like. In some embodiments, the close proximity of the first nuclear spin and the second nuclear spin gives rise to a strong J-coupling between the first nuclear spin and the nuclear spin, allowing for efficient transfer of magnetization between the first nuclear spin and the second nuclear spin.

[092] In some embodiments, target magnetization associated with the target molecule (or a metabolic derivative of the target molecule) is selectively preserved by utilizing the J-coupling network of the target molecule (or the metabolic derivative of the target molecule).

[093] At 520, the subject or the portion of the subject is subjected to a magnetic field in an MRI machine. In some embodiments, the magnetic field has any average magnitude described herein with respect to step 120 of FIG. 1. In some embodiments, subjecting the subject or the portion of the subject to the magnetic field generates target magnetization in the first nuclear spin of the target molecule or the metabolic derivative of the target molecule and generates nontarget magnetization other MRI-active molecules in the subject or the portion of the subject.

[094] In some embodiments, step 520 is performed a predetermined time period after step 510. In some embodiments, performing step 520 a predetermined time period after step 510 permits the subject or the portion of the subject to display a biological response to the target molecule. That is, in some embodiments, during the predetermined time period, the target molecule is permitted to obtain a biological response in the subject or the portion of the subject. In some embodiments, the biological response is indicative of a biological status of the subject or the portion of the subject. In some embodiments, the predetermined time period is any predetermined time described herein with respect to FIG. 1.

[095] At 530, one or more pulse sequences are performed to preserve the target magnetization and to suppress the non-target magnetization. In some embodiments, the one or more pulse sequences are performed on the first nuclear spin and the second nuclear spin. That is, in some embodiments, the one or more pulse sequences are applied to perform spin dynamics operations on the first nuclear spin and the second nuclear spin. In some embodiments, the one or more pulse sequences utilize a J-coupling network of the target molecule (or the metabolic derivative of the target molecule) to selectively preserve the target magnetization. For instance, in some embodiments, the one or more pulse sequences utilize the J-coupling between the first nuclear spin and the second nuclear spin to selectively preserve the target magnetization. In some embodiments, the one or more pulse sequences are configured to reduce the non-target magnetization by a factor of at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000,

2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, or more, a factor of at most about 100,000, 90,000, 80,000, 70,000, 60,000, 50,000, 40,000, 30,000, 20,000, 10,000, 9,000, 8,000, 7,000, 6,000, 5,000, 4,000, 3,000, 2,000, 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 100, or less, or a factor that is within a range defined by any two of the preceding values. In some embodiments, the reduction in the non-target magnetization is in comparison to the non-target magnetization that would be expected in the absence of the pulse sequences described herein. In some embodiments, the one or more pulses sequences are configured to suppress the non-target magnetization such that most or substantially all of the remaining magnetization comprises target magnetization. In some embodiments, the one or more pulse sequences correspond to the one or more pulse sequences described herein with respect to step 610 of FIG. 6. In some embodiments, the one or more pulse sequences correspond to the one or more first pulse sequences and the one or more second pulse sequences described herein with respect to steps 710 and 730, respectively, of FIG. 7.

[096] At 540, one or more MRI images of the target molecule or the metabolic derivative of the target molecule in the subject or the portion of the subject are obtained. In some embodiments, the one or more MRI images correspond to the one or more MRI images described herein with respect to step 620 of FIG. 6. In some embodiments, the one or more MRI images correspond to the one or more first MRI images, the one or more second MRI images, and the one or more background- subtracted MRI images described herein with respect to steps 720, 740, and 750, respectively, of FIG. 7.

[097] At 550, a biological status of the subject or the portion of the subject is determined based on the one or more MRI images.

Methods for acquiring MRI images by preserving target magnetization and suppressing non-target magnetization via magnetization transfer or heteronuclear correlations using J-coupled nuclear spins of different types

[098] FIG. 6 depicts an exemplary method 600 for acquiring MRI images by preserving target magnetization and suppressing non-target magnetization via magnetization transfer or heteronuclear correlations using J-coupled nuclear spins of different types. At 610, one or more pulse sequences are performed to transfer the target magnetization from the first nuclear spin to magnetization on the second nuclear spin or to convert the target magnetization into a heteronuclear correlation between the first nuclear spin and the second nuclear spin, suppress the non-target magnetization, and regenerate the target magnetization from the magnetization on the second nuclear spin or from the heteronuclear correlation between the first nuclear spin and the second nuclear spin. For instance, in some embodiments, one or more pulse sequences are performed to transfer target magnetization from protons to carbon- 13 magnetization, suppress non-target magnetization, and regenerate the target magnetization from the carbon- 13 magnetization. As another example, in some embodiments, the one or more pulse sequences are performed to transfer target magnetization from protons to a heteronuclear correlation between protons and carbon- 13 nuclei, suppress non-target magnetization, and regenerate the target magnetization from the heteronuclear correlation between the protons and the carbon- 13 nuclei. In some embodiments, the one or more pulse sequences utilize the J-coupling network of the target molecule or the metabolic derivative of the target molecule. For instance, in some embodiments, the one or more pulse sequences utilize the J-coupling between the first nuclear spin and the second nuclear spin to transfer the target magnetization to magnetization on the second nuclear spin, suppress the non-target magnetization, and regenerate the target magnetization. In some embodiments, the one or more pulse sequences comprise one or more PulsePol pulse sequences, one or more gradient spoiling sequences, one or more radiofrequency (RF) spoiling pulse sequences, one or more magnetization transfer pulse sequences, one or more insensitive nuclei enhanced by polarization transfer (INEPT) pulse sequences, one or more carb on- 13 -proton heteronuclear correlation pulse sequences, or any combination thereof. In some embodiments, preserving the target magnetization in the magnetization on the second nuclear spin prevents the target magnetization from being affected by spin operations (such as gradient spoiling or RF spoiling) that affects the non-preserved, non-target magnetization. Then, the non-target magnetization can be suppressed using spin operations such as the gradient spoiling or RF spoiling. Finally, the target magnetization can be regenerated from the magnetization on the second nuclear spin. Thus, following step 610, most or substantially all of the remaining magnetization is target magnetization.

[099] At 620, one or more MRI images of the target molecule or the metabolic derivative of the target molecule in the subject or the portion of the subject are obtained.

Methods for acquiring MRI images by preserving target magnetization and suppressing non-target magnetization via phase operations using J-coupled nuclear spins of different types

[0100] FIG. 7 depicts an exemplary method 700 for acquiring MRI images by preserving target magnetization and suppressing non-target magnetization via phase operations using J-coupled nuclear spins of different types. At 710, one or more first pulse sequences are performed to preserve the target magnetization and to generate first target magnetization having a first phase. In some embodiments, the first phase is selected such that the first target magnetization is aligned parallel to the magnetic field. In some embodiments, the one or more first pulse sequences utilize the J-coupling network of the target molecule or the metabolic derivative of the target molecule. In some embodiments, the J-coupling between the first nuclear spin and the second nuclear spin is used to preserve the target magnetization and to generate the first target magnetization. For instance, in some embodiments, the J-coupling between the first nuclear spin and the second nuclear spin is used to transfer the target magnetization to magnetization on the second nuclear spin, suppress the non-target magnetization, and generate the first target magnetization from the magnetization on the second nuclear spin, as described herein with respect to step 610 of FIG. 6. In some embodiments, the one or more first pulse sequences comprise one or more PulsePol pulse sequences, one or more gradient spoiling sequences, one or more radio-frequency (RF) spoiling pulse sequences, one or more polarization transfer pulse sequences, one or more insensitive nuclei enhanced by polarization transfer (INEPT) pulse sequences, one or more carbon- 13 -proton heteronuclear correlation pulse sequences, or any combination thereof.

[0101] At 720, one or more first MRI images of the target molecule or the metabolic derivative of the target molecule in the subject or the portion of the subject are obtained based on the first target magnetization.

[0102] At 730, one or more second pulse sequences are performed to the target magnetization and to generate second target magnetization having a second phase. In some embodiments, the second phase is different from the first phase. In some embodiments, the second phase is selected such that the second target magnetization is aligned anti-parallel to the magnetic field. In some embodiments, the second phase is selected such that the second target magnetization is aligned parallel to the magnetic field. In some embodiments, the one or more second pulse sequences utilize the J-coupling network of the target molecule or the metabolic derivative of the target molecule. In some embodiments, the J-coupling between the first nuclear spin and the second nuclear spin is used to preserve the target magnetization and to generate the second target magnetization. For instance, in some embodiments, the J-coupling between the first nuclear spin and the second nuclear spin is used to transfer the target magnetization to magnetization on the second nuclear spin, suppress the non-target magnetization, and generate the second target magnetization from the magnetization on the second nuclear spin, as described herein with respect to step 610 of FIG. 6. In some embodiments, the one or more second pulse sequences comprise one or more PulsePol pulse sequences, one or more gradient spoiling sequences, one or more radio-frequency (RF) spoiling pulse sequences, one or more polarization transfer pulse sequences, one or more insensitive nuclei enhanced by polarization transfer (INEPT) pulse sequences, one or more carbon- 13 -proton heteronuclear correlation pulse sequences, or any combination thereof.

[0103] At 740, one or more second MRI images of the target molecule or the metabolic derivative of the target molecule in the subject or the portion of the subject are obtained based on the second target magnetization.

[0104] At 750, the one or more second MRI images are subtracted from the one or more first MRI images to obtain one or more background-subtracted MRI images. In some embodiments, the one or more background-subtracted MRI images comprise suppressed MRI signals from the non-target magnetization. That is, assuming that the first and second pulse sequences are substantially identical (with the exception of the first and second phases imparted to the target magnetization), the MRI signals resulting from the non-target magnetization will be substantially identical in the first and second MRI images. However, the MRI signals resulting from the target magnetization will be out of phase (e.g., 180 degrees out of phase when the first and second target magnetizations are aligned parallel and anti-parallel to the magnetic field, respectively). Thus, when the one or more second MRI images are subtracted from the one or more first MRI images, the MRI signals resulting from the non-target magnetization will substantially cancel out. On the other hand, the MRI signals resulting from the target magnetization will remain. For instance, when the first and second target magnetizations are aligned parallel and anti-parallel to the magnetic field, the remaining MRI signal will be approximately twice as strong as the MRI signals resulting from the target magnetization in the one or more first MRI images or the one or more second MRI images.

[0105] Although FIG. 7 describes method 700 as utilizing first target magnetization that is aligned parallel to the magnetic field and second target magnetization that is aligned antiparallel to the magnetic field, the disclosure is not intended to be so limiting. The first and second phases may have any possible values, so long as they are different. For instance, the first phase may be chosen such that the first target magnetization is aligned anti-parallel to the magnetic field and the second target magnetization is aligned parallel to the magnetic field. The second phase may be different from the first phase by at least about 10 degrees, 20 degrees, 30 degrees, 40 degrees, 50 degrees, 60 degrees, 70 degrees, 80 degrees, 90 degrees, 100 degrees, 110 degrees, 120 degrees, 130 degrees, 140 degrees, 150 degrees, 160 degrees, 170 degrees, 180 degrees, or more, at most about 180 degrees, 170 degrees, 160 degrees, 150 degrees, 140 degrees, 130 degrees, 120 degrees, 110 degrees, 100 degrees, 90 degrees, 80 degrees, 70 degrees, 60 degrees, 50 degrees, 40 degrees, 30 degrees, 20 degrees, 10 degrees, or less, or by an angle that is within a range defined by any two of the preceding values.

Methods for acquiring MRI images based on multiple relatively low-abundance molecules using J-coupled nuclear spins of different types

[0106] FIG. 8 depicts an exemplary method 800 for acquiring MRI images based on multiple relatively low-abundance molecules using J-coupled nuclear spins of different types. At 810, a target molecule is provided to a subject or a portion of a subject. In some embodiments, the target molecule comprises a first nuclear spin and a second nuclear spin. In some embodiments, the second nuclear spin is of a different spin species from the first nuclear spin. That is, in some embodiments, the first nuclear spin and the second nuclear spin are different nuclear isotopes. For instance, in some embodiments, the first nuclear spin and the second nuclear spin comprise any first nuclear spin and second nuclear spin, respectively, described herein with respect to FIG. 5. In some embodiments, the first nuclear spin and the second nuclear spin are coupled by an electron-mediated J-coupling. In some embodiments, the J-coupling between the first nuclear spin and the second nuclear spin is any J-coupling described herein with respect to FIG. 5. In some embodiments, the first nuclear spin is chemically bonded (e.g., covalently bonded) to the second nuclear spin (i.e., the first nuclear spin and the second nuclear spin are located one chemical bond away from one another), as described herein with respect to FIG. 5. In some embodiments, the close proximity of the first nuclear spin and the second nuclear spin gives rise to a strong J-coupling between the first nuclear spin and the second nuclear spin, allowing for efficient transfer of magnetization between the first nuclear spin and the second nuclear spin, as described herein with respect to FIG. 5. In some embodiments, target magnetization associated with the target molecule is selectively preserved by utilizing the J-coupling network of the target molecule.

[0107] At 820, at least a portion of the target molecule is permitted to be converted to a derivative molecule (e.g., any metabolic derivative of the target molecule described herein). In some embodiments, the derivative molecule comprises the first nuclear spin and the second nuclear spin. In some embodiments, the first nuclear spin and the second nuclear spin are coupled by an electron-mediated J-coupling. In some embodiments, the J-coupling between the first nuclear spin and the second nuclear spin is any J-coupling described herein with respect to step 510 of FIG. 5.

[0108] In some embodiments, derivative target magnetization associated with the derivative molecule is selectively preserved (e.g., by transferring target magnetization on the first nuclear spin to magnetization on the second nuclear spin, suppressing non-target magnetization, and regenerating the target magnetization from the magnetization on the second nuclear spin) by utilizing the J-coupling network of the derivative molecule.

[0109] At 830, the subject or the portion of the subject is subjected to a magnetic field in an MRI machine. In some embodiments, the magnetic field has any average magnitude described herein with respect to step 120 of FIG. 1. In some embodiments, subjecting the subject or the portion of the subject to the magnetic field generates target magnetization in the target molecule, generates derivative target magnetization in the derivative molecule, and generates non-target magnetization in other MRI-active molecules in the subject or the portion of the subject.

[0110] In some embodiments, step 830 is performed a predetermined time period after step 810. In some embodiments, performing step 830 a predetermined time period after step 810 permits the subject or the portion of the subject to display a biological response to the target molecule. That is, in some embodiments, during the predetermined time period, the target molecule is permitted to obtain a biological response in the subject or the portion of the subject. In some embodiments, the biological response is indicative of a biological status of the subject or the portion of the subject. In some embodiments, the predetermined time period comprises any predetermined time period described herein with respect to step 120 of FIG. 1.

[OHl] At 840, one or more pulse sequences are performed to preserve the target magnetization and to suppress the non-target magnetization and the derivative target magnetization. In some embodiments, the one or more pulse sequences utilize a J-coupling network of the target molecule to selectively preserve the target magnetization. In some embodiments, the one or more pulse sequences are configured to suppress any amount of non-target magnetization and derivative target magnetization described herein with respect to step 530 of FIG. 5. In some embodiments, the one or more pulses sequences are configured to suppress the non-target magnetization and the derivative target magnetization such that most or substantially all of the remaining magnetization comprises target magnetization. In some embodiments, the one or more pulse sequences correspond to the one or more pulse sequences described herein with respect to step 610 of FIG. 6. In some embodiments, the one or more pulse sequences correspond to the one or more first pulse sequences and the one or more second pulse sequences described herein with respect to steps 710 and 730, respectively, of FIG. 7.

[0112] At 850, one or more MRI images of the target molecule in the subject or the portion of the subject are obtained. In some embodiments, the one or more MRI images correspond to the one or more MRI images described herein with respect to step 620 of FIG. 6. In some embodiments, the one or more MRI images correspond to the one or more first MRI images, the one or more second MRI images, and the one or more background- subtracted MRI images described herein with respect to steps 720, 740, and 750, respectively, of FIG. 7.

[0113] At 860, one or more pulse sequences are performed to preserve the derivative target magnetization and to suppress the non-target magnetization and the target magnetization. In some embodiments, the one or more pulse sequences utilize a J-coupling network of the derivative molecule to selectively preserve the derivative target magnetization. In some embodiments, the one or more pulse sequences are configured to suppress any amount of nontarget magnetization and target magnetization described herein with respect to step 530 of FIG. 5. In some embodiments, the one or more pulses sequences are configured to suppress the nontarget magnetization and the target magnetization such that most or substantially all of the remaining magnetization comprises derivative target magnetization. In some embodiments, the one or more pulse sequences correspond to the one or more pulse sequences described herein with respect to step 610 of FIG. 6. In some embodiments, the one or more pulse sequences correspond to the one or more first pulse sequences and the one or more second pulse sequences described herein with respect to steps 710 and 730, respectively, of FIG. 7.

[0114] At 870, one or more MRI images of the derivative molecule in the subject or the portion of the subject are obtained. In some embodiments, the one or more MRI images correspond to the one or more MRI images described herein with respect to step 620 of FIG. 6. In some embodiments, the one or more MRI images correspond to the one or more first MRI images, the one or more second MRI images, and the one or more background- subtracted MRI images described herein with respect to steps 720, 740, and 750, respectively, of FIG. 7.

[0115] At 880, a biological status of the subj ect or the portion of the subj ect is determined based on the one or more MRI images of the target molecule and the one or more MRI images of the derivative molecule.

EXAMPLES

Example 1: Determination of Necrosis Status via MRI Imaging of Fumarate or Malate

[0116] The systems and methods described herein may be used to determine whether a subject or a portion of the subject is displaying necrosis. When administered to a subject or a portion of the subject that is not displaying necrosis, fumarate has difficulty getting into cellular mitochondria and is thus not readily metabolized. However, when necrosis is present, cells eject numerous macromolecules, including the enzymes that convert fumarate to its metabolic derivatives (e.g., malate). Thus, fumarate can be administered as a target molecule, a wait of a predetermined time period can be performed, and one or more MRI images can be obtained using the systems and methods described herein. For instance, one or more MRI images of fumarate or malate can be obtained. If the one or more MRI images show only fumarate, this is an indication that the subject or the portion of the subject is not displaying necrosis. However, any voxels in the one or more MRI images that contain substances other than fumarate (e.g., malate) may be indicative of necrosis. The fumarate (and malate) can be carbon-13 labeled in order to break the symmetry between the first proton spin and the second proton spin in fumarate, thus enabling transitions between the singlet state and triplet states of the first proton and second proton spins. Alternatively, the fumarate (and malate) can comprise a J-coupled proton spin and an isotopically enriched carbon-13 spin, thus enabling magnetization transfer from target magnetization to carbon- 13 magnetization, suppression of non-target magnetization, and magnetization transfer from carbon- 13 magnetization to target magnetization.

Example 2: Determination of Aerobic Status via MRI Imaging of Glucose, Glutamine, or Glutamate

[0117] The systems and methods described herein may be used to determine whether a subject or a portion of the subject is undergoing aerobic or anaerobic metabolism. When cells metabolize glucose, different metabolic derivatives are formed depending on whether the cells are engaging in aerobic or anaerobic metabolism. Thus, glucose can be administered as a target molecule, a wait of a predetermined time period can be performed, and one or more MRI images can be obtained using the systems and methods described herein. For instance, one or more MRI images of glucose, glutamine, or glutamate can be obtained. Based on the relative strength of the resulting MRI signals, an aerobic status or an anaerobic status of the subject or the portion of the subject may be assigned on a voxel -by- voxel basis. The glucose (and glutamine and glutamate) can be carbon- 13 labeled or deuterium labeled to help to distinguish exogenously administered glucose (and glutamine and glutamate) from endogenous glucose (and glutamine and glutamate) that may already be present within cells. Alternatively, the glucose (and glutamine and glutamate) can comprise a I-coupled proton spin and an isotopically enriched carbon- 13 spin, thus enabling magnetization transfer from target magnetization to carbon- 13 magnetization, suppression of non-target magnetization, and magnetization transfer from carbon- 13 magnetization to target magnetization.

[0118] In another embodiment, endogenous glucose and its metabolic derivatives glutamine and glutamate can be used as imaging targets. An advantage of this embodiment is that no external agent needs to be injected or digested.

Example 3: Pulse sequences for suppression of NMR signals associated with water and with protons that are not chemically bonded to carbon-13 nuclei

[0119] FIG. 9A depicts a first exemplary pulse sequence for suppressing magnetization associated with water and protons that are not chemically bonded to carbon-13 nuclei. The pulse sequence depicted in FIG. 9 A imparts three major spin dynamics operations. First, magnetization associated with a proton that is chemically bonded to a carbon-13 nucleus is converted into a carb on- 13 -proton correlation. This first operation is depicted in the pulse upper left pulse block in FIG. 9A, which shows a series of three proton NMR pulses and a carbon-13 NMR pulse. This first pulse block stores proton magnetization as a carbon- 13 -proton correlation only if the proton is chemically bonded to a carbon- 13 nucleus and has a J-coupling of approximately JCH with such a carbon-13. Proton magnetization associated with water or with protons that are not chemically bonded to such carbon- 13 nuclei (i.e., protons that are chemically bonded to carbon- 12 nuclei) is not stored. Second, non-stored magnetization is dephased using a gradient spoiling pulse sequence (depicted as G in FIG. 9A). Use of the gradient spoiling pulse sequence effectively suppresses magnetization associated with water and with protons that are not chemically bonded to a carbon- 13 nucleus. Third, the carbon-13- proton correlations are converted back into proton magnetization. This third operation is depicted in the upper right pulse block in FIG. 9A, which shows a series of three proton NMR pulses and a carbon- 13 NMR pulse. Since the non-stored magnetization was suppressed during the second operation, essentially all remaining magnetization is non associated only with protons that are chemically bonded to a carbon- 13 nucleus. Finally, an NMR spectrum can be collected using standard spectroscopic techniques. The pulse sequence depicted in FIG. 9A may also be modified using standard spectroscopic techniques such as phase cycling, cleanup gradients, and the like.

[0120] FIG. 9B depicts a second exemplary pulse sequence for suppressing magnetization associated with water and protons that are not chemically bonded to carbon- 13 nuclei. In comparison with the pulse sequence depicted in FIG. 9A, the pulse sequence depicted in FIG. 9B converts proton magnetization into 13C magnetization rather than converting proton magnetization into a carb on- 13 -proton correlation.

Example 4: Suppression of NMR signals associated with water and with protons that are not chemically bonded to carbon-13 nuclei

[0121] FIG. 10 depicts exemplary NMR spectrum of water, fumarate, and malic acid obtained in the absence of (top spectrum) and using (bottom spectrum) the pulse sequence depicted in FIG. 9 A. Briefly, a 90%/10% mixture of water and deuterated water was prepared. 400 millimolar (mM) disodium fumarate and 400 mM malic acid were added to the water- deuterated water mixture. The fumarate and malic acid were prepared with carbon- 13 at its natural isotopic abundance (i.e., approximately 99% carbon-12 and approximately 1% carbon- 13 at each carbon in the fumarate and malic acid). NMR. spectra was acquired using a 9.4 tesla (T) NMR spectrometer. As shown in FIG. 10, the NMR spectrum is dominated by water in the absence of the pulse sequence depicted in FIG. 9A. Additionally, NMR peaks associated with various protons (chemical bonded to both carbon-13 and to carbon-12) are present. Table 1 shows the integrated NMR signals obtained in the absence of the pulse sequence depicted in FIG. 9A.

Table 1 : Integrated NMR signals associated with water, fumarate, and malic acid in the absence of the pulse sequence depicted in FIG. 9A

[0122] By contrast, the NMR signal associated with water is greatly decreased (by a factor of approximately 3,000) when using the pulse sequence depicted in FIG. 9A. Additionally, the NMR peaks associated with protons chemically bonded to carbon- 12 are reduced (by a factor of approximately 5,000, though many such peaks were difficult to integrate due to the low remaining signals). By contrast, the NMR signals associated with protons chemically bonded to carbon-13 were reduced by no more than 50%. Table 2 shows the integrated NMR signals obtained using the pulse sequence depicted in FIG. 9A.

Table 2: Integrated NMR signals associated with water, fumarate, and malic acid using the pulse sequence depicted in FIG. 9A

reduce NMR signals associated with water and with protons that are not chemically bonded to carbon-13 nuclei. While the pulse sequence depicted in FIG. 9A does not suppress the water signal completely, additional techniques may be useful for further suppressing the water signal. For instance, water suppression pulse blocks such as MEGA-PRESS may be combined with the pulse sequence depicted in FIG. 9A to further reduce the water signal. Alternatively or in combination, carbon- 13 labeled molecules (such as carbon- 13 labeled fumarate or carbon- 13 labeled malic acid) may be utilized to increase the NMR signals associated with protons that are chemically bonded to carbon-13 nuclei by a factor of up to 100. Using such techniques, the NMR signals associated with protons that are chemically bonded to carbon- 13 nuclei may become the dominant signals in the NMR spectrum. Such techniques may thus create clean enough NMR spectra to permit acquisition of MRI images whose signal originates almost entirely from non-suppressed magnetization.

Example 5: Monitoring conversion of fumarate to malate by suppressing NMR signals associated with water and with protons that are not chemically bonded to carbon-13 nuclei

[0124] FIG. 11 depicts an exemplary time-series graph showing the conversion of fumarate to malic acid. Briefly, a 90%/10% mixture of water and deuterated water was prepared. 25 mM carbon-13 labeled disodium fumarate, 70 mM glutamine, and 140 mM pyruvate were added to the water-deuterated water mixture. The glutamine and pyruvate were prepared with carbon- 13 at its natural isotopic abundance (i.e., approximately 99% carbon-12 and approximately 1% carbon- 13 at each carbon in the fumarate). NMR spectra were acquired at each time point using a 9.4 T NMR spectrometer. At each time point, the pulse sequence depicted in FIG. 9A was utilized to suppress NMR signals associated with water and with protons that are not chemically bonded to carbon- 13 nuclei, as described herein. The suppression of such signals allowed the NMR signals associated with fumarate ¹³C-3 and fumarate ¹³C-4 to be easily identified and integrated. While the pulse sequence depicted in FIG. 9A does not suppress the protons signals associated with water, glutamine ¹³C, or pyruvate ¹³C completely, carbon- 13 labeling of fumarate means that the protons signals associated with fumarate ¹³C dominate over those associated with glutamine ¹³C and pyruvate ¹³C. As discussed above, additional techniques may be useful for further suppressing such signals. For instance, water suppression pulse blocks such as MEGA-PRESS may be combined with the pulse sequence depicted in FIG. 9A to further reduce the water signal. Using such techniques, the proton signals associated with fumarate ¹³C (and its metabolic derivative malate ¹³C) may become the dominant signal in the NMR spectrum. Such techniques may thus create clean enough NMR spectra to permit acquisition of MRI images whose signal originates almost entirely from fumarate and malate.

Example 6: Suppression of water signals for MRI imaging

[0125] FIG. 12 depicts exemplary NMR spectra showing the suppression of water signals in a mixture of water of carbon- 13 labeled acetone in preparation for MRI imaging. NMR spectra of a phantom containing water and 1-¹³C acetone were obtained using a Bruker preclinical 11.7 T imaging system. FIG. 12 depicts an exemplary proton NMR spectrum associated with the imaging phantom with (heavily suppressed water peak) and without (includes a large water peak) the pulse sequence depicted in FIG. 9A. As shown in FIG. 12, the water peak is highly suppressed using the methods described herein. While the pulse sequence depicted in FIG. 9A does not suppress the water completely, additional techniques may be useful for further suppressing the water signal. For instance, water suppression pulse blocks such as MEGAPRESS may be combined with the pulse sequence depicted in FIG. 9A to further reduce the water signal. Using such techniques, the water NMR signals may be suppressed enough to permit acquisition of MRI images whose signal originates almost entirely from acetone or other molecules described herein (such as fumarate, malic acid, malate, and the like).

RECITATION OF EMBODIMENTS

[0126] Embodiment 1. A method comprising:

(a) identifying a target molecule in a subject or a portion of the subject, the target molecule comprising a first nuclear spin and a second nuclear spin, wherein the first nuclear spin and the second nuclear spin are of the same spin species, wherein a J- coupling between the first nuclear spin and the second nuclear spin is at least 1 hertz (Hz), and wherein: (i) a chemical shift difference between the first nuclear spin and the second nuclear spin is between 0.001 parts per million (ppm) and 1 ppm or

(ii) the target molecule further comprises a third nuclear spin, wherein a J- coupling between the third nuclear spin and the first nuclear spin is different than a J-coupling between the third nuclear spin and the second nuclear spin;

(b) subjecting the subject or the portion of the subject to a magnetic field in a magnetic resonance imaging (MRI) machine to induce a target magnetization in the target molecule or a metabolic derivative of the target molecule and to induce a non-target magnetization in other MRI-active molecules in the subject or the portion of the subject;

(c) performing one or more pulse sequences to preserve the target magnetization and to suppress the non-target magnetization by utilizing a J-coupling network of the target molecule or the metabolic derivative of the target molecule to selectively preserve the target magnetization;

(d) obtaining one or more MRI images of the target molecule or the metabolic derivative of the target molecule in the subject or the portion of the subject; and

(e) determining a biological status of the subject or the portion of the subject based on the one or more MRI images.

[0127] Embodiment 2. The method of Embodiment 1, wherein the first nuclear spin and the second nuclear spin each comprise a proton nuclear spin, wherein the first nuclear spin and the second nuclear spin each comprise a carbon- 13 nuclear spin or an isotopically enriched carbon- 13 nuclear spin, or wherein the first nuclear spin and the second nuclear spin each comprise a nitrogen-15 nuclear spin or an isotopically enriched carbon-13 nuclear spin.

[0128] Embodiment 3. The method of Embodiment 1 or 2, wherein the target molecule comprises at least one member selected from the group consisting of: a biologically active molecule, an antibody, a drug, an antibody-drug conjugate, a radionuclide, a radionuclide-drug conjugate, fumarate, glucose, and any carbon- 13 labeled, nitrogen- 15 labeled, partially deuterated, or fully deuterated analog thereof.

[0129] Embodiment 4. The method of any one of Embodiments 1-3, wherein the metabolic derivative comprises at least one member selected from the group consisting of: a metabolic derivative of a biologically active molecule, a metabolic derivative of a drug, a metabolic derivative of an antibody-drug conjugate, a metabolic derivative of a radionuclide-drug conjugate, malate, glutamate, and any carbon- 13 labeled, nitrogen- 15 labeled, partially deuterated, or fully deuterated analog thereof.

[0130] Embodiment 5. The method of any one of Embodiments 1-4, wherein the non-target magnetization comprises magnetization associated with water or fat in the subject or the portion of the subject.

[0131] Embodiment 6. The method of any one of Embodiments 1-5, wherein the biological status comprises at least one member selected from the group consisting of: a biological reaction of the subject or the portion of the subject to a biologically active molecule or a metabolic derivative of a biologically active molecule, a biological reaction of the subject or the portion of the subject to an antibody, a biological reaction of the subject or the portion of the subject to a drug or a metabolic derivative of a drug, a biological reaction of the subject or the portion of the subject to an antibody-drug conjugate or a metabolic derivative of an antibody-drug conjugate, a biological reaction of the subject or the portion of the subject to a radionuclide, a biological reaction of the subject or the portion of the subject to a radionuclide-drug conjugate or a metabolic derivative of a radionuclide-drug conjugate, a presence or absence of necrosis in the subject or the portion of the subject, an aerobic status of the subject or the portion of the subject, and an anaerobic status of the subject or the portion of the subject.

[0132] Embodiment 7. The method of any one of Embodiments 1-6, wherein the one or more pulse sequences are configured to reduce the non-target magnetization in the other MRI-active molecules by a factor of at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000,

3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, or 100,000.

[0133] Embodiment 8. The method of any one of Embodiments 1-7, wherein (b) is performed a predetermined time period after (a).

[0134] Embodiment 9. The method of Embodiment 8, wherein the predetermined time period is at least 1 minute (min), 2 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 1 hour (h), 2 h, or 5 h.

[0135] Embodiment 10. The method of Embodiment 8 or 9, wherein, during the predetermined time period, the target molecule interacts with the subject to obtain a biological response that is indicative of the biological status of the subject or the portion of the subject.

[0136] Embodiment 11. The method of any one of Embodiments 1-10, wherein the target molecule is endogenous to the subject or the portion of the subject.

[0137] Embodiment 12. The method of any one of Embodiments 1-10, wherein the target molecule is exogenous to the subject or the portion of the subject.

[0138] Embodiment 13. The method of Embodiment 12, further comprising, prior to (b), administering the target molecule to the subject or the portion of the subject.

[0139] Embodiment 14. The method of Embodiment 13, wherein the target molecule comprises partially or fully deuterated or carbon- 13 labeled fumarate, wherein the metabolic derivative of the target molecule comprises partially or fully deuterated or carbon- 13 labeled malate, and wherein the biological status of the subject or the portion of the subject comprises a presence or absence of necrosis in the subject or the portion of the subject.

[0140] Embodiment 15. The method of Embodiment 13, wherein the target molecule comprises partially or fully deuterated or carbon- 13 labeled glucose, wherein the metabolic derivative of the target molecule comprises partially or fully deuterated or carbon- 13 labeled glutamine or partially or fully deuterated or carbon- 13 labeled glutamate, and wherein the biological status of the subject or the portion of the subject comprises an aerobic status of the subject or the portion of the subject or an anaerobic status of the subject or the portion of the subject.

[0141] Embodiment 16. The method of any one of Embodiments 1-15, wherein the one or more pulse sequences preserve the target magnetization in a singlet state, suppress the non-target magnetization, and regenerate the target magnetization from the singlet state.

[0142] Embodiment 17. The method of any one of Embodiments 1-16, wherein the one or more pulse sequences comprise one or more PulsePol pulse sequences.

[0143] Embodiment 18. The method of any one of Embodiments 1-17, wherein the one or more pulse sequences comprise one or more gradient spoiling pulse sequences.

[0144] Embodiment 19. The method of any one of Embodiments 1-18, wherein the one or more pulse sequences comprise one or more radio-frequency (RF) spoiling pulse sequences.

[0145] Embodiment 20. A method comprising:

(a) identifying a target molecule in a subject or a portion of the subject;

(c) performing one or more pulse sequences to preserve the target magnetization in a singlet state, suppress the non-target magnetization, and regenerate the target magnetization from the singlet state utilizing a J-coupling network of the target molecule or the metabolic derivative of the target molecule;

[0146] Embodiment 21. The method of Embodiment 20, wherein the target molecule comprises at least one member selected from the group consisting of: a biologically active molecule, an antibody, a drug, an antibody-drug conjugate, a radionuclide, a radionuclide-drug conjugate, fumarate, glucose, and any carbon- 13 labeled, nitrogen- 15 labeled, partially deuterated, or fully deuterated analog thereof.

[0147] Embodiment 22. The method of Embodiment 20 or 21, wherein the metabolic derivative comprises at least one member selected from the group consisting of: a metabolic derivative of a biologically active molecule, a metabolic derivative of a drug, a metabolic derivative of an antibody-drug conjugate, a metabolic derivative of a radionuclide-drug conjugate, malate, glutamate, and any carbon- 13 labeled, nitrogen- 15 labeled, partially deuterated, or fully deuterated analog thereof.

[0148] Embodiment 23. The method of any one of Embodiments 20-22, wherein the non-target magnetization comprises magnetization associated with water or fat in the subject or the portion of the subject.

[0149] Embodiment 24. The method of any one of Embodiments 20-23, wherein the biological status comprises at least one member selected from the group consisting of: a biological reaction of the subject or the portion of the subject to a biologically active molecule or a metabolic derivative of a biologically active molecule, a biological reaction of the subject or the portion of the subject to an antibody, a biological reaction of the subject or the portion of the subject to a drug or a metabolic derivative of a drug, a biological reaction of the subject or the portion of the subject to an antibody-drug conjugate or a metabolic derivative of an antibody-drug conjugate, a biological reaction of the subject or the portion of the subject to a radionuclide, a biological reaction of the subject or the portion of the subject to a radionuclide-drug conjugate or a metabolic derivative of a radionuclide-drug conjugate, a presence or absence of necrosis in the subject or the portion of the subject, an aerobic status of the subject or the portion of the subject, and an anaerobic status of the subject or the portion of the subject.

[0150] Embodiment 25. The method of any one of Embodiments 20-24, wherein the one or more pulse sequences are configured to reduce the non-target magnetization in the other MRI- active molecules by a factor of at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, or 100,000.

[0151] Embodiment 26. The method of any one of Embodiments 20-25, wherein (b) is performed a predetermined time period after (a).

[0152] Embodiment 27. The method of Embodiment 26, wherein the predetermined time period is at least 1 minute (min), 2 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 1 hour (h), 2 h, or 5 h.

[0153] Embodiment 28. The method of Embodiment 26 or 27, wherein, during the predetermined time period, the target molecule interacts with the subject to obtain a biological response that is indicative of the biological status of the subject or the portion of the subject.

[0154] Embodiment 29. The method of any one of Embodiments 20-28, wherein the target molecule is endogenous to the subject or the portion of the subject.

[0155] Embodiment 30. The method of any one of Embodiments 20-28, wherein the target molecule is exogenous to the subject or the portion of the subject.

[0156] Embodiment 31. The method of Embodiment 30, further comprising, prior to (b), administering the target molecule to the subject or the portion of the subject.

[0157] Embodiment 32. The method of Embodiment 31, wherein the target molecule comprises partially or fully deuterated or carbon- 13 labeled fumarate, wherein the metabolic derivative of the target molecule comprises partially or fully deuterated or carbon- 13 labeled malate, and wherein the biological status of the subject or the portion of the subject comprises a presence or absence of necrosis in the subject or the portion of the subject.

[0158] Embodiment 33. The method of Embodiment 31, wherein the target molecule comprises partially or fully deuterated or carbon- 13 labeled glucose, wherein the metabolic derivative of the target molecule comprises partially or fully deuterated or carbon- 13 labeled glutamine or partially or fully deuterated or carbon- 13 labeled glutamate, and wherein the biological status of the subject or the portion of the subject comprises an aerobic status of the subject or the portion of the subject or an anaerobic status of the subject or the portion of the subject.

[0159] Embodiment 34. The method of any one of Embodiments 20-33, wherein the one or more pulse sequences comprise one or more PulsePol pulse sequences.

[0160] Embodiment 35. The method of any one of Embodiments 20-34, wherein the one or more pulse sequences comprise one or more gradient spoiling pulse sequences.

[0161] Embodiment 36. The method of any one of Embodiments 20-35, wherein the one or more pulse sequences comprise one or more radio-frequency (RF) spoiling pulse sequences.

[0162] Embodiment 37. The method of any one of Embodiments 20-36, wherein the target molecule comprises a first nuclear spin and a second nuclear spin, wherein the first nuclear spin and the second nuclear spin are of the same spin species, wherein a J-coupling between the first nuclear spin and the second nuclear spin is at least 1 hertz (Hz), and wherein:

(i) a chemical shift difference between the first nuclear spin and the second nuclear spin is between 0.001 parts per million (ppm) and 1 ppm or

(ii) the target molecule further comprises a third nuclear spin, wherein a J-coupling between the third nuclear spin and the first nuclear spin is different than a J-coupling between the third nuclear spin and the second nuclear spin.

[0163] Embodiment 38. The method of Embodiment 37, wherein the first nuclear spin and the second nuclear spin each comprise a proton nuclear spin, wherein the first nuclear spin and the second nuclear spin each comprise a carbon- 13 nuclear spin or an isotopically enriched carbon- 13 nuclear spin, or wherein the first nuclear spin and the second nuclear spin each comprise a nitrogen-15 nuclear spin or an isotopically enriched carbon-13 nuclear spin.

[0164] Embodiment 39. A method comprising:

(a) identifying a target molecule in a subject or a portion of the subject;

(b) subjecting the subject or the portion of the subject to a magnetic field in a magnetic resonance imaging (MRI) machine to induce a target magnetization in the target molecule or a metabolic derivative of the target molecule and to induce a non-target magnetization in other MRI-active molecules;

(c) performing one or more first pulse sequences to preserve the target magnetization in a singlet state and generate first target magnetization having a first phase from the singlet state utilizing a J-coupling network of the target molecule or the metabolic derivative of the target molecule;

(d) obtaining one or more first MRI images of the target molecule or the metabolic derivative of the target molecule in the subject or the portion of the subject based on the first target magnetization;

(e) performing one or more second pulse sequences to preserve the target magnetization in a singlet state and generate second target magnetization having a second phase from the singlet state utilizing a J-coupling network of the target molecule or the metabolic derivative of the target molecule, wherein the second phase is different from the first phase;

(f) obtaining one or more second MRI images of the target molecule or the metabolic derivative of the target molecule in the subject or the portion of the subject based on the second target magnetization; (g) subtracting the one or more second MRI images from the one or more first MRI images to thereby obtain one or more background- subtracted MRI images comprising suppressed MRI signals from the non-target magnetization; and

(h) determining a biological status of the subject or the portion of the subject based on the one or more background- subtracted MRI images.

[0165] Embodiment 40. The method of Embodiment 39, wherein the target molecule comprises at least one member selected from the group consisting of: a biologically active molecule, an antibody, a drug, an antibody-drug conjugate, a radionuclide, a radionuclide-drug conjugate, fumarate, glucose, and any carbon- 13 labeled, nitrogen- 15 labeled, partially deuterated, or fully deuterated analog thereof.

[0166] Embodiment 41. The method of Embodiment 39 or 40, wherein the metabolic derivative comprises at least one member selected from the group consisting of: a metabolic derivative of a biologically active molecule, a metabolic derivative of a drug, a metabolic derivative of an antibody-drug conjugate, a metabolic derivative of a radionuclide-drug conjugate, malate, glutamate, and any carbon- 13 labeled, nitrogen- 15 labeled, partially deuterated, or fully deuterated analog thereof.

[0167] Embodiment 42. The method of any one of Embodiments 39-41, wherein the non-target magnetization comprises magnetization associated with water or fat in the subject or the portion of the subject.

[0168] Embodiment 43. The method of any one of Embodiments 39-42, wherein the biological status comprises at least one member selected from the group consisting of: a biological reaction of the subject or the portion of the subject to a biologically active molecule or a metabolic derivative of a biologically active molecule, a biological reaction of the subject or the portion of the subject to an antibody, a biological reaction of the subject or the portion of the subject to a drug or a metabolic derivative of a drug, a biological reaction of the subject or the portion of the subject to an antibody-drug conjugate or a metabolic derivative of an antibody-drug conjugate, a biological reaction of the subject or the portion of the subject to a radionuclide, a biological reaction of the subject or the portion of the subject to a radionuclide-drug conjugate or a metabolic derivative of a radionuclide-drug conjugate, a presence or absence of necrosis in the subject or the portion of the subject, an aerobic status of the subject or the portion of the subject, and an anaerobic status of the subject or the portion of the subject.

[0169] Embodiment 44. The method of any one of Embodiments 39-43, wherein the one or more first or second pulse sequences are configured to reduce the non-target magnetization in the other MRI-active molecules by a factor of at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, or 100,000.

[0170] Embodiment 45. The method of any one of Embodiments 39-44, wherein (b) is performed a predetermined time period after (a).

[0171] Embodiment 46. The method of Embodiment 45, wherein the predetermined time period is at least 1 minute (min), 2 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 1 hour (h), 2 h, or 5 h.

[0172] Embodiment 47. The method of Embodiment 45 or 46, wherein, during the predetermined time period, the target molecule interacts with the subject to obtain a biological response that is indicative of the biological status of the subject or the portion of the subject.

[0173] Embodiment 48. The method of any one of Embodiments 39-47, wherein the target molecule is endogenous to the subject or the portion of the subject.

[0174] Embodiment 49. The method of any one of Embodiments 39-47, wherein the target molecule is exogenous to the subject or the portion of the subject.

[0175] Embodiment 50. The method of Embodiment 49, further comprising, prior to (b), administering the target molecule to the subject or the portion of the subject. [0176] Embodiment 51. The method of Embodiment 49, wherein the target molecule comprises partially or fully deuterated or carbon- 13 labeled fumarate, wherein the metabolic derivative of the target molecule comprises partially or fully deuterated or carbon- 13 labeled malate, and wherein the biological status of the subject or the portion of the subject comprises a presence or absence of necrosis in the subject or the portion of the subject.

[0177] Embodiment 52. The method of Embodiment 49, wherein the target molecule comprises partially or fully deuterated or carbon- 13 labeled glucose, wherein the metabolic derivative of the target molecule comprises partially or fully deuterated or carbon- 13 labeled glutamine or partially or fully deuterated or carbon- 13 labeled glutamate, and wherein the biological status of the subject or the portion of the subject comprises an aerobic status of the subject or the portion of the subject or an anaerobic status of the subject or the portion of the subject.

[0178] Embodiment 53. The method of any one of Embodiments 39-52, wherein the one or more first or second pulse sequences comprise one or more PulsePol pulse sequences.

[0179] Embodiment 54. The method of any one of Embodiments 39-53, wherein the one or more first or second pulse sequences comprise one or more gradient spoiling pulse sequences.

[0180] Embodiment 55. The method of any one of Embodiments 39-54, wherein the one or more first or second pulse sequences comprise one or more radio-frequency (RF) spoiling pulse sequences.

[0181] Embodiment 56. The method of any one of Embodiments 39-55, wherein the target molecule comprises a first nuclear spin and a second nuclear spin, wherein the first nuclear spin and the second nuclear spin are of the same spin species, wherein a J-coupling between the first nuclear spin and the second nuclear spin is at least 1 hertz (Hz), and wherein:

[0182] Embodiment 57. The method of Embodiment 56, wherein the first nuclear spin and the second nuclear spin each comprise a proton nuclear spin, wherein the first nuclear spin and the second nuclear spin each comprise a carbon- 13 nuclear spin or an isotopically enriched carbon- 13 nuclear spin, or wherein the first nuclear spin and the second nuclear spin each comprise a nitrogen-15 nuclear spin or an isotopically enriched carbon-13 nuclear spin.

[0183] Embodiment 58. The method of any one of Embodiments 39-57, wherein the second phase is 180 degrees different from the first phase.

[0184] Embodiment 59. The method of any one of Embodiments 39-58, wherein the first target magnetization is aligned parallel to the magnetic field and wherein the second target magnetization is aligned antiparallel to the magnetic field or wherein the first target magnetization is aligned antiparallel to the magnetic field and the second target magnetization is aligned parallel to the magnetic field.

[0185] Embodiment 60. A method comprising:

(a) identifying a target molecule in a subject or a portion of the subject, the target molecule comprising with a first nuclear spin and a second nuclear spin, wherein the first nuclear spin and the second nuclear spin are the of the same spin species, wherein a J-coupling between the first nuclear spin and the second nuclear spin is at least 1 hertz (Hz), and wherein:

(ii) the target molecule further comprises a third nuclear spin, wherein a J- coupling between the third nuclear spin and the first nuclear spin is different than a J-coupling between the third nuclear spin and the second nuclear spin; (b) permitting at least a portion of the target molecule to be converted to a derivative molecule in the subject or the portion of the subject, the derivative molecule comprising at least a first nuclear spin and a second nuclear spin, wherein the first nuclear spin and the second nuclear spin are of the same spin species, wherein a J- coupling between of the first nuclear spin and the second nuclear spin is at least 1 Hz, and wherein:

(i) a chemical shift difference between the first nuclear spin and the second nuclear spin is between 0.001 ppm and 1 ppm or

(ii) the derivative molecule comprises a third nuclear spin, wherein a J-coupling between the third nuclear spin and the first nuclear spin is different than a J- coupling between the third nuclear spin and the second nuclear spin;

(c) subjecting the subject or the portion of the subject to a magnetic field in a magnetic resonance imaging (MRI) machine to induce a target magnetization in the target molecule and a derivative target magnetization in the derivative molecule and to induce a non-target magnetization in other MRI-active molecules in the subject or the portion of the subject;

(d) performing one or more first pulse sequences to preserve the target magnetization and to suppress the non-target magnetization and the derivative target magnetization by utilizing a J-coupling network of the target molecule to selectively preserve the target magnetization;

(e) obtaining one or more MRI images of the target molecule in the subject or the portion of the subject;

(f) performing one or more second pulse sequences to preserve the derivative target magnetization and to suppress the non-target magnetization and the target magnetization by utilizing a J-coupling network of the derivative molecule to selectively preserve the derivative target magnetization;

(g) obtaining one or more MRI images of the derivative molecule in the subject or the portion of the subject; and

(h) determining a biological status of the subject or the portion of the subject based on the one or more MRI images of the target molecule and the one or more MRI images of the derivative molecule.

[0186] Embodiment 61. The method of Embodiment 60, wherein the first nuclear spin and the second nuclear spin each comprise a proton nuclear spin, wherein the first nuclear spin and the second nuclear spin each comprise a carbon- 13 nuclear spin or an isotopically enriched carbon- 13 nuclear spin, or wherein the first nuclear spin and the second nuclear spin each comprise a nitrogen-15 nuclear spin or an isotopically enriched carbon-13 nuclear spin.

[0187] Embodiment 62. The method of Embodiment 60 or 61, wherein the target molecule comprises at least one member selected from the group consisting of: a biologically active molecule, an antibody, a drug, an antibody-drug conjugate, a radionuclide, a radionuclide-drug conjugate, fumarate, glucose, and any carbon- 13 labeled, nitrogen- 15 labeled, partially deuterated, or fully deuterated analog thereof.

[0188] Embodiment 63. The method of any one of Embodiments 60-62, wherein the derivative molecule comprises at least one member selected from the group consisting of: a metabolic derivative of a biologically active molecule, a metabolic derivative of a drug, a metabolic derivative of an antibody-drug conjugate, a metabolic derivative of a radionuclide-drug conjugate, malate, glutamate, and any carbon- 13 labeled, nitrogen- 15 labeled, partially deuterated, or fully deuterated analog thereof.

[0189] Embodiment 64. The method of any one of Embodiments 60-63, wherein the non-target magnetization comprises magnetization associated with water or fat in the subject or the portion of the subject. [0190] Embodiment 65. The method of any one of Embodiments 60-64, wherein the biological status comprises at least one member selected from the group consisting of: a biological reaction of the subject or the portion of the subject to a biologically active molecule or a metabolic derivative of a biologically active molecule, a biological reaction of the subject or the portion of the subject to an antibody, a biological reaction of the subject or the portion of the subject to a drug or a metabolic derivative of a drug, a biological reaction of the subject or the portion of the subject to an antibody-drug conjugate or a metabolic derivative of an antibody-drug conjugate, a biological reaction of the subject or the portion of the subject to a radionuclide, a biological reaction of the subject or the portion of the subject to a radionuclide-drug conjugate or a metabolic derivative of a radionuclide-drug conjugate, a presence or absence of necrosis in the subject or the portion of the subject, an aerobic status of the subject or the portion of the subject, and an anaerobic status of the subject or the portion of the subject.

[0191] Embodiment 66. The method of any one of Embodiments 60-65, wherein the one or more first or second pulse sequences are configured to reduce the non-target magnetization in the other MRI-active molecules by a factor of at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, or 100,000.

[0192] Embodiment 67. The method of any one of Embodiments 60-66, wherein (c) is performed a predetermined time period after (a).

[0193] Embodiment 68. The method of Embodiment 67, wherein the predetermined time period is at least 1 minute (min), 2 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 1 hour (h), 2 h, or 5 h.

[0194] Embodiment 69. The method of Embodiment 67 or 68, wherein, during the predetermined time period, the target molecule interacts with the subject to obtain a biological response that is indicative of the biological status of the subject or the portion of the subject. [0195] Embodiment 70. The method of any one of Embodiments 60-69, wherein the target molecule is endogenous to the subject or the portion of the subject.

[0196] Embodiment 71. The method of any one of Embodiments 60-69, wherein the target molecule is exogenous to the subject or the portion of the subject.

[0197] Embodiment 72. The method of Embodiment 71, further comprising, prior to (c), administering the target molecule to the subject or the portion of the subject.

[0198] Embodiment 73. The method of Embodiment 72, wherein the target molecule comprises partially or fully deuterated or carbon- 13 labeled fumarate, wherein the derivative molecule comprises partially or fully deuterated or carbon-13 labeled malate, and wherein the biological status of the subject or the portion of the subject comprises a presence or absence of necrosis in the subject or the portion of the subject.

[0199] Embodiment 74. The method of Embodiment 72, wherein the target molecule comprises partially or fully deuterated or carbon- 13 labeled glucose, wherein the derivative molecule comprises partially or fully deuterated or carbon-13 labeled glutamine or partially or fully deuterated or carbon- 13 labeled glutamate, and wherein the biological status of the subject or the portion of the subject comprises an aerobic status of the subject or the portion of the subject or an anaerobic status of the subject or the portion of the subject.

[0200] Embodiment 75. The method of any one of Embodiments 60-74, wherein the one or more first pulse sequences preserve the target magnetization in a first singlet state, suppress the non-target magnetization and the derivative target magnetization, and regenerate the target magnetization from the first singlet state or wherein the one or more second pulse sequences preserve the derivative target magnetization in a second singlet state, suppress the non-target magnetization and the target magnetization, and regenerate the derivative target magnetization from the second singlet state.

[0201] Embodiment 76. The method of any one of Embodiments 60-75, wherein the one or more first or second pulse sequences comprise one or more PulsePol pulse sequences.

[0202] Embodiment 77. The method of any one of Embodiments 60-76, wherein the one or more first or second pulse sequences comprise one or more gradient spoiling pulse sequences. [0203] Embodiment 78. The method of any one of Embodiments 60-77, wherein the one or more first or second pulse sequences comprise one or more radio-frequency (RF) spoiling pulse sequences.

[0204] Embodiment 79. A method comprising:

(a) providing a target molecule to a subject or a portion of the subject, the target molecule comprising a first nuclear spin and a second nuclear spin of a different spin species from the first nuclear spin, wherein a J-coupling between the first nuclear spin and the second nuclear spin is at least 10 hertz (Hz);

(c) performing one or more pulse sequences on the first nuclear spin and the second nuclear spin to preserve the target magnetization and to suppress the non-target magnetization by utilizing the J-coupling between the first nuclear spin and the second nuclear spin of the target molecule or the metabolic derivative of the target molecule to selectively preserve the target magnetization;

(e) determining a biological status of the subject or the portion of the subject based on the one or more MRI images. [0205] Embodiment 80. The method of Embodiment 79, wherein the first nuclear spin comprises a proton nuclear spin and wherein the second nuclear spin comprises a carbon- 13 nuclear spin, an isotopically enriched carbon-13 nuclear spin, a nitrogen-14 nuclear spin, a nitrogen-15 nuclear spin, an isotopically enriched nitrogen-15 nuclear spin, an oxygen-17 nuclear spin, an isotopically enriched nuclear spin, a fluorine-19 nuclear spin, or a phosphorus- 31 nuclear spin.

[0206] Embodiment 81. The method of Embodiment 79 or 80, wherein the target molecule comprises at least one member selected from the group consisting of: a biologically active molecule, an antibody, a drug, an antibody-drug conjugate, a radionuclide, a radionuclide-drug conjugate, fumarate, glucose, and any carbon-13 labeled, nitrogen-15 labeled, oxygen-17 labeled, partially deuterated, or fully deuterated analog thereof.

[0207] Embodiment 82. The method of any one of Embodiments 79-81, wherein the metabolic derivative comprises at least one member selected from the group consisting of: a metabolic derivative of a biologically active molecule, a metabolic derivative of a drug, a metabolic derivative of an antibody-drug conjugate, a metabolic derivative of a radionuclide-drug conjugate, malate, glutamate, and any carbon-13 labeled, nitrogen-15 labeled, oxygen-17 labeled, partially deuterated, or fully deuterated analog thereof.

[0208] Embodiment 83. The method of any one of Embodiments 79-82, wherein the non-target magnetization comprises magnetization associated with water or fat in the subject or the portion of the subject.

[0209] Embodiment 84. The method of any one of Embodiments 79-83, wherein the biological status comprises at least one member selected from the group consisting of: a biological reaction of the subject or the portion of the subject to a biologically active molecule or a metabolic derivative of a biologically active molecule, a biological reaction of the subject or the portion of the subject to an antibody, a biological reaction of the subject or the portion of the subject to a drug or a metabolic derivative of a drug, a biological reaction of the subject or the portion of the subject to an antibody-drug conjugate or a metabolic derivative of an antibody-drug conjugate, a biological reaction of the subject or the portion of the subject to a radionuclide, a biological reaction of the subject or the portion of the subject to a radionuclide-drug conjugate or a metabolic derivative of a radionuclide-drug conjugate, a presence or absence of necrosis in the subject or the portion of the subject, an aerobic status of the subject or the portion of the subject, and an anaerobic status of the subject or the portion of the subject.

[0210] Embodiment 85. The method of any one of Embodiments 79-84, wherein the one or more pulse sequences are configured to reduce the non-target magnetization in the other MRI- active molecules by a factor of at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, or 100,000.

[0211] Embodiment 86. The method of any one of Embodiments 79-85, wherein (b) is performed a predetermined time period after (a).

[0212] Embodiment 87. The method of Embodiment 86, wherein the predetermined time period is at least 1 minute (min), 2 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 1 hour (h), 2 h, or 5 h.

[0213] Embodiment 88. The method of Embodiment 86 or 87, wherein, during the predetermined time period, the target molecule interacts with the subject to obtain a biological response that is indicative of the biological status of the subject or the portion of the subject.

[0214] Embodiment 89. The method of any one of Embodiments 79-88, wherein the target molecule comprises carbon-13 labeled fumarate, wherein the metabolic derivative of the target molecule comprises carbon- 13 labeled malate, and wherein the biological status of the subject or the portion of the subject comprises a presence or absence of necrosis in the subject or the portion of the subject. [0215] Embodiment 90. The method of any one of Embodiments 79-89, wherein the first nuclear spin is chemically bonded to the second nuclear spin.

[0216] Embodiment 91. The method of any one of Embodiments 79-90, wherein the one or more pulse sequences preserve the target magnetization and suppress the non-target magnetization by transferring the target magnetization to magnetization on the second nuclear spin or by converting the target magnetization into a heteronuclear correlation between the first nuclear spin and the second nuclear spin, suppressing the non-target magnetization, and regenerating the target magnetization from the magnetization on the second nuclear spin or the heteronuclear correlation between the first nuclear spin and the second nuclear spin.

[0217] Embodiment 92. The method of any one of Embodiments 79-91, wherein the one or more pulse sequences comprise one or more PulsePol pulse sequences.

[0218] Embodiment 93. The method of any one of Embodiments 79-92, wherein the one or more pulse sequences comprise one or more gradient spoiling pulse sequences.

[0219] Embodiment 94. The method of any one of Embodiments 79-93, wherein the one or more pulse sequences comprise one or more radio-frequency (RF) spoiling pulse sequences.

[0220] Embodiment 95. The method of any one of Embodiments 79-94, wherein the one or more pulse sequences comprise one or more polarization transfer pulse sequences.

[0221] Embodiment 96. The method of any one of Embodiments 79-95, wherein the one or more pulse sequences comprise one or more insensitive nuclei enhanced by polarization transfer (INEPT) pulse sequences.

[0222] Embodiment 97. The method of any one of Embodiments 79-94, wherein the one or more pulse sequences comprise one or more carb on- 13 -proton heteronuclear correlation pulse sequences.

[0223] Embodiment 98. A method comprising:

(a) providing a target molecule to a subject or a portion of the subject, the target molecule comprising a first nuclear spin and a second nuclear spin of a different spin species from the first nuclear spin;

(b) subjecting the subject or the portion of the subject to a magnetic field in a magnetic resonance imaging (MRI) machine to induce a target magnetization in the first nuclear spin of the target molecule or a metabolic derivative of the target molecule and to induce a non-target magnetization in other MRI-active molecules in the subject or the portion of the subject;

(c) performing one or more pulse sequences on the first nuclear spin and the second nuclear spin to transfer the target magnetization to magnetization on the second nuclear spin or to convert the target magnetization into a heteronuclear correlation between the first nuclear spin and the second nuclear spin, suppress the non-target magnetization, and regenerate the target magnetization from the magnetization on the second nuclear spin or the heteronuclear correlation between the first nuclear spin and the second nuclear spin utilizing a J-coupling between the first nuclear spin and the second nuclear spin of the target molecule or the metabolic derivative of the target molecule;

[0224] Embodiment 99. The method of Embodiment 98, wherein the first nuclear spin comprises a proton nuclear spin and wherein the second nuclear spin comprises a carbon- 13 nuclear spin, an isotopically enriched carbon-13 nuclear spin, a nitrogen-14 nuclear spin, a nitrogen-15 nuclear spin, an isotopically enriched nitrogen-15 nuclear spin, an oxygen-17 nuclear spin, an isotopically enriched nuclear spin, a fluorine-19 nuclear spin, or a phosphorus- 31 nuclear spin.

[0225] Embodiment 100. The method of Embodiment 98 or 99, wherein the target molecule comprises at least one member selected from the group consisting of: a biologically active molecule, an antibody, a drug, an antibody-drug conjugate, a radionuclide, a radionuclide-drug conjugate, fumarate, glucose, and any carbon-13 labeled, nitrogen-15 labeled, oxygen-17 labeled, partially deuterated, or fully deuterated analog thereof.

[0226] Embodiment 101. The method of any one of Embodiments 98-100, wherein the metabolic derivative comprises at least one member selected from the group consisting of: a metabolic derivative of a biologically active molecule, a metabolic derivative of a drug, a metabolic derivative of an antibody-drug conjugate, a metabolic derivative of a radionuclidedrug conjugate, malate, glutamate, and any carbon- 13 labeled, nitrogen- 15 labeled, oxygen- 17 labeled, partially deuterated, or fully deuterated analog thereof.

[0227] Embodiment 102. The method of any one of Embodiments 98-101, wherein the nontarget magnetization comprises magnetization associated with water or fat in the subject or the portion of the subject.

[0228] Embodiment 103. The method of any one of Embodiments 98-102, wherein the biological status comprises at least one member selected from the group consisting of: a biological reaction of the subject or the portion of the subject to a biologically active molecule or a metabolic derivative of a biologically active molecule, a biological reaction of the subject or the portion of the subject to an antibody, a biological reaction of the subject or the portion of the subject to a drug or a metabolic derivative of a drug, a biological reaction of the subject or the portion of the subject to an antibody-drug conjugate or a metabolic derivative of an antibody-drug conjugate, a biological reaction of the subject or the portion of the subject to a radionuclide, a biological reaction of the subject or the portion of the subject to a radionuclidedrug conjugate or a metabolic derivative of a radionuclide-drug conjugate, a presence or absence of necrosis in the subject or the portion of the subject, an aerobic status of the subject or the portion of the subject, and an anaerobic status of the subject or the portion of the subject. [0229] Embodiment 104. The method of any one of Embodiments 98-103, wherein the one or more pulse sequences are configured to reduce the non-target magnetization in the other MRI- active molecules by a factor of at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, or 100,000.

[0230] Embodiment 105. The method of any one of Embodiments 98-104, wherein (b) is performed a predetermined time period after (a).

[0231] Embodiment 106. The method of Embodiment 105, wherein the predetermined time period is at least 1 minute (min), 2 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 1 hour (h), 2 h, or 5 h.

[0232] Embodiment 107. The method of Embodiment 105 or 106, wherein, during the predetermined time period, the target molecule interacts with the subject to obtain a biological response that is indicative of the biological status of the subject or the portion of the subject.

[0233] Embodiment 108. The method of any one of Embodiments 98-107, wherein the target molecule comprises carbon-13 labeled fumarate, wherein the metabolic derivative of the target molecule comprises carbon- 13 labeled malate, and wherein the biological status of the subject or the portion of the subject comprises a presence or absence of necrosis in the subject or the portion of the subject.

[0234] Embodiment 109. The method of any one of Embodiments 98-108, wherein the first nuclear spin is chemically bonded to the second nuclear spin.

[0235] Embodiment 110. The method of any one of Embodiments 98-109, wherein the one or more pulse sequences comprise one or more PulsePol pulse sequences.

[0236] Embodiment 111. The method of any one of Embodiments 98-110, wherein the one or more pulse sequences comprise one or more gradient spoiling pulse sequences.

[0237] Embodiment 112. The method of any one of Embodiments 98-111, wherein the one or more pulse sequences comprise one or more radio-frequency (RF) spoiling pulse sequences.

[0238] Embodiment 113. The method of any one of Embodiments 98-112, wherein the one or more pulse sequences comprise one or more polarization transfer pulse sequences.

[0239] Embodiment 114. The method of any one of Embodiments 98-113, wherein the one or more pulse sequences comprise one or more insensitive nuclei enhanced by polarization transfer (INEPT) pulse sequences.

[0240] Embodiment 115. The method of any one of Embodiments 98-113, wherein the one or more pulse sequences comprise one or more carb on- 13 -proton heteronuclear correlation pulse sequences.

[0241] Embodiment 116. The method of any one of Embodiments 98-114, wherein a J-coupling between the first nuclear spin and the second nuclear spin is at least 10 hertz (Hz).

[0242] Embodiment 117. A method comprising:

(c) performing one or more first pulse sequences on the first nuclear spin and the second nuclear spin to preserve the target magnetization and generate first target magnetization having a first phase utilizing a J-coupling between the first nuclear spin and the second nuclear spin of the target molecule or the metabolic derivative of the target molecule;

(e) performing one or more second pulse sequences to preserve the target magnetization and generate second target magnetization having a second phase utilizing the I- coupling between the first nuclear spin and the second nuclear spin of the target molecule or the metabolic derivative of the target molecule, wherein the second phase is different from the first phase;

(f) obtaining one or more second MRI images of the target molecule or the metabolic derivative of the target molecule in the subject or the portion of the subject based on the second target magnetization;

(g) subtracting the one or more second MRI images from the one or more first MRI images to thereby obtain one or more background- subtracted MRI images comprising suppressed MRI signals from the non-target magnetization; and

[0243] Embodiment 118. The method of Embodiment 117, wherein the first nuclear spin comprises a proton nuclear spin and wherein the second nuclear spin comprises a carbon- 13 nuclear spin, an isotopically enriched carbon-13 nuclear spin, a nitrogen-14 nuclear spin, a nitrogen-15 nuclear spin, an isotopically enriched nitrogen-15 nuclear spin, an oxygen-17 nuclear spin, an isotopically enriched nuclear spin, a fluorine-19 nuclear spin, or a phosphorus- 31 nuclear spin.

[0244] Embodiment 119. The method of Embodiment 117 or 118, wherein the target molecule comprises at least one member selected from the group consisting of a biologically active molecule, an antibody, a drug, an antibody-drug conjugate, a radionuclide, a radionuclide-drug conjugate, fumarate, glucose, and any carbon-13 labeled, nitrogen-15 labeled, oxygen-17 labeled, partially deuterated, or fully deuterated analog thereof.

[0245] Embodiment 120. The method of any one of Embodiments 117-119, wherein the metabolic derivative comprises at least one member selected from the group consisting of: a metabolic derivative of a biologically active molecule, a metabolic derivative of a drug, a metabolic derivative of an antibody-drug conjugate, a metabolic derivative of a radionuclidedrug conjugate, malate, glutamate, and any carbon- 13 labeled, nitrogen- 15 labeled, oxygen- 17 labeled, partially deuterated, or fully deuterated analog thereof.

[0246] Embodiment 121. The method of any one of Embodiments 117-120, wherein the nontarget magnetization comprises magnetization associated with water or fat in the subject or the portion of the subject.

[0247] Embodiment 122. The method of any one of Embodiments 117-121, wherein the biological status comprises at least one member selected from the group consisting of: a biological reaction of the subject or the portion of the subject to a biologically active molecule or a metabolic derivative of a biologically active molecule, a biological reaction of the subject or the portion of the subject to an antibody, a biological reaction of the subject or the portion of the subject to a drug or a metabolic derivative of a drug, a biological reaction of the subject or the portion of the subject to an antibody-drug conjugate or a metabolic derivative of an antibody-drug conjugate, a biological reaction of the subject or the portion of the subject to a radionuclide, a biological reaction of the subject or the portion of the subject to a radionuclidedrug conjugate or a metabolic derivative of a radionuclide-drug conjugate, a presence or absence of necrosis in the subject or the portion of the subject, an aerobic status of the subject or the portion of the subject, and an anaerobic status of the subject or the portion of the subject. [0248] Embodiment 123. The method of any one of Embodiments 117-122, wherein the one or more first or second pulse sequences are configured to reduce the non-target magnetization in the other MRI-active molecules by a factor of at least 100, 200, 300, 400, 500, 600, 700, 800,

900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, or 100,000.

[0249] Embodiment 124. The method of any one of Embodiments 117-123, wherein (b) is performed a predetermined time period after (a).

[0250] Embodiment 125. The method of Embodiment 124, wherein the predetermined time period is at least 1 minute (min), 2 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 1 hour (h), 2 h, or 5 h.

[0251] Embodiment 126. The method of Embodiment 124 or 125, wherein, during the predetermined time period, the target molecule interacts with the subject to obtain a biological response that is indicative of the biological status of the subject or the portion of the subject.

[0252] Embodiment 127. The method of any one of Embodiments 117-126, wherein the target molecule comprises carbon-13 labeled fumarate, wherein the metabolic derivative of the target molecule comprises carbon- 13 labeled malate, and wherein the biological status of the subject or the portion of the subject comprises a presence or absence of necrosis in the subject or the portion of the subject.

[0253] Embodiment 128. The method of any one of Embodiments 117-127, wherein the first nuclear spin is chemically bonded to the second nuclear spin.

[0254] Embodiment 129. The method of any one of Embodiments 117-128, wherein the one or more first or second pulse sequences comprise one or more PulsePol pulse sequences.

[0255] Embodiment 130. The method of any one of Embodiments 117-129, wherein the one or more first or second pulse sequences comprise one or more gradient spoiling pulse sequences. [0256] Embodiment 131. The method of any one of Embodiments 117-130, wherein the one or more first or second pulse sequences comprise one or more radio-frequency (RF) spoiling pulse sequences.

[0257] Embodiment 132. The method of any one of Embodiments 117-131, wherein the one or more first or second pulse sequences comprise one or more polarization transfer pulse sequences.

[0258] Embodiment 133. The method of any one of Embodiments 117-132, wherein the one or more first or second pulse sequences comprise one or more insensitive nuclei enhanced by polarization transfer (INEPT) pulse sequences.

[0259] Embodiment 134. The method of any one of Embodiments 117-131, wherein the one or more pulse sequences comprise one or more carb on- 13 -proton heteronuclear correlation pulse sequences.

[0260] Embodiment 135. The method of any one of Embodiments 117-134, the J-coupling between the first nuclear spin and the second nuclear spin is at least 10 hertz (Hz).

[0261] Embodiment 136. The method of any one of Embodiments 117-135, wherein the second phase is 180 degrees different from the first phase.

[0262] Embodiment 137. The method of any one of Embodiments 117-136, wherein the first target magnetization is aligned parallel to the magnetic field and wherein the second target magnetization is aligned antiparallel to the magnetic field or wherein the first target magnetization is aligned antiparallel to the magnetic field and the second target magnetization is aligned parallel to the magnetic field.

[0263] Embodiment 138. A method comprising:

(b) permitting at least a portion of the target molecule to be converted to a derivative molecule in the subject or the portion of the subject, the derivative molecule comprising the first nuclear spin and the second nuclear spin, wherein a J-coupling between the first nuclear spin and the second nuclear spin is at least 10 Hz;

(d) performing one or more first pulse sequences to preserve the target magnetization and to suppress the non-target magnetization and the derivative target magnetization by utilizing the J-coupling between the first nuclear spin and the second nuclear spin to selectively preserve the target magnetization;

(f) performing one or more second pulse sequences to preserve the derivative target magnetization and to suppress the non-target magnetization and the target magnetization by utilizing the J-coupling between the first nuclear spin and the second nuclear spin to selectively preserve the derivative target magnetization;

[0264] Embodiment 139. The method of Embodiment 138, wherein the first nuclear spin comprises a proton nuclear spin and wherein the second nuclear spin comprises a carbon- 13 nuclear spin, an isotopically enriched carbon-13 nuclear spin, a nitrogen-14 nuclear spin, a nitrogen-15 nuclear spin, an isotopically enriched nitrogen-15 nuclear spin, an oxygen-17 nuclear spin, an isotopically enriched nuclear spin, a fluorine-19 nuclear spin, or a phosphorus- 31 nuclear spin.

[0265] Embodiment 140. The method of Embodiment 138 or 139, wherein the target molecule comprises at least one member selected from the group consisting of: a biologically active molecule, an antibody, a drug, an antibody-drug conjugate, a radionuclide, a radionuclide-drug conjugate, fumarate, glucose, and any carbon-13 labeled, nitrogen-15 labeled, oxygen-17 labeled, partially deuterated, or fully deuterated analog thereof.

[0266] Embodiment 141. The method of any one of Embodiments 138-140, wherein the derivative molecule comprises at least one member selected from the group consisting of: a metabolic derivative of a biologically active molecule, a metabolic derivative of a drug, a metabolic derivative of an antibody-drug conjugate, a metabolic derivative of a radionuclidedrug conjugate, malate, glutamate, and any carbon- 13 labeled, nitrogen- 15 labeled, oxygen- 17 labeled, partially deuterated, or fully deuterated analog thereof.

[0267] Embodiment 142. The method of any one of Embodiments 138-141, wherein the nontarget magnetization comprises magnetization associated with water or fat in the subject or the portion of the subject.

[0268] Embodiment 143. The method of any one of Embodiments 138-142, wherein the biological status comprises at least one member selected from the group consisting of: a biological reaction of the subject or the portion of the subject to a biologically active molecule or a metabolic derivative of a biologically active molecule, a biological reaction of the subject or the portion of the subject to an antibody, a biological reaction of the subject or the portion of the subject to a drug or a metabolic derivative of a drug, a biological reaction of the subject or the portion of the subject to an antibody-drug conjugate or a metabolic derivative of an antibody-drug conjugate, a biological reaction of the subject or the portion of the subject to a radionuclide, a biological reaction of the subject or the portion of the subject to a radionuclidedrug conjugate or a metabolic derivative of a radionuclide-drug conjugate, a presence or absence of necrosis in the subject or the portion of the subject, an aerobic status of the subject or the portion of the subject, and an anaerobic status of the subject or the portion of the subject. [0269] Embodiment 144. The method of any one of Embodiments 138-143, wherein the one or more first or second pulse sequences are configured to reduce the non-target magnetization in the other MRI-active molecules by a factor of at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, or 100,000.

[0270] Embodiment 145. The method of any one of Embodiments 138-144, wherein (c) is performed a predetermined time period after (a).

[0271] Embodiment 146. The method of Embodiment 145, wherein the predetermined time period is at least 1 minute (min), 2 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 1 hour (h), 2 h, or 5 h.

[0272] Embodiment 147. The method of Embodiment 145 or 146, wherein, during the predetermined time period, the target molecule interacts with the subject to obtain a biological response that is indicative of the biological status of the subject or the portion of the subject.

[0273] Embodiment 148. The method of any one of Embodiments 138-147, wherein the target molecule comprises carbon- 13 labeled fumarate, wherein the derivative molecule comprises carbon- 13 labeled malate, and wherein the biological status of the subject or the portion of the subject comprises a presence or absence of necrosis in the subject or the portion of the subject. [0274] Embodiment 149. The method of any one of Embodiments 138-148, wherein the first nuclear spin is chemically bonded to the second nuclear spin.

[0275] Embodiment 150. The method of any one of Embodiments 138-149, wherein the one or more first or second pulse sequences comprise one or more PulsePol pulse sequences.

[0276] Embodiment 151. The method of any one of Embodiments 138-150, wherein the one or more first or second pulse sequences comprise one or more gradient spoiling pulse sequences. [0277] Embodiment 152. The method of any one of Embodiments 138-151, wherein the one or more first or second pulse sequences comprise one or more radio-frequency (RF) spoiling pulse sequences.

[0278] Embodiment 153. The method of any one of Embodiments 138-152, wherein the one or more first or second pulse sequences comprise one or more polarization transfer pulse sequences.

[0279] Embodiment 154. The method of any one of Embodiments 138-153, wherein the one or more first or second pulse sequences comprise one or more insensitive nuclei enhanced by polarization transfer (INEPT) pulse sequences.

[0280] Embodiment 155. The method of any one of Embodiments 138-152, wherein the one or more pulse sequences comprise one or more carb on- 13 -proton heteronuclear correlation pulse sequences.

[0281] Embodiment 156. A method comprising:

(a) identifying a target molecule in a subject or a portion of the subject:

(c) performing one or more pulse sequences to preserve the target magnetization and to suppress the non-target magnetization by a factor of at least 1,000 utilizing a J- coupling network of the target molecule or the metabolic derivative of the target molecule to selectively preserve the target magnetization;

[0282] Embodiment 157. The method of Embodiment 156, wherein the target molecule comprises at least one member selected from the group consisting of: a biologically active molecule, an antibody, a drug, an antibody-drug conjugate, a radionuclide, a radionuclide-drug conjugate, fumarate, glucose, and any carbon- 13 labeled, nitrogen- 15 labeled, partially deuterated, or fully deuterated analog thereof.

[0283] Embodiment 158. The method of Embodiment 156 or 157, wherein the metabolic derivative comprises at least one member selected from the group consisting of: a metabolic derivative of a biologically active molecule, a metabolic derivative of a drug, a metabolic derivative of an antibody-drug conjugate, a metabolic derivative of a radionuclide-drug conjugate, malate, glutamate, and any carbon- 13 labeled, nitrogen- 15 labeled, partially deuterated, or fully deuterated analog thereof.

[0284] Embodiment 159. The method of any one of Embodiments 156-158, wherein the nontarget magnetization comprises magnetization associated with water or fat in the subject or the portion of the subject.

[0285] Embodiment 160. The method of any one of Embodiments 156-159, wherein the biological status comprises at least one member selected from the group consisting of: a biological reaction of the subject or the portion of the subject to a biologically active molecule or a metabolic derivative of a biologically active molecule, a biological reaction of the subject or the portion of the subject to an antibody, a biological reaction of the subject or the portion of the subject to a drug or a metabolic derivative of a drug, a biological reaction of the subject or the portion of the subject to an antibody-drug conjugate or a metabolic derivative of an antibody-drug conjugate, a biological reaction of the subject or the portion of the subject to a radionuclide, a biological reaction of the subject or the portion of the subject to a radionuclidedrug conjugate or a metabolic derivative of a radionuclide-drug conjugate, a presence or absence of necrosis in the subject or the portion of the subject, an aerobic status of the subject or the portion of the subject, and an anaerobic status of the subject or the portion of the subject. [0286] Embodiment 161. The method of any one of Embodiments 156-160, wherein the one or more pulse sequences are configured to reduce the non-target magnetization in the other MRI- active molecules by a factor of at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, or 100,000.

[0287] Embodiment 162. The method of any one of Embodiments 156-161, wherein (b) is performed a predetermined time period after (a).

[0288] Embodiment 163. The method of Embodiment 162, wherein the predetermined time period is at least 1 minute (min), 2 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 1 hour (h), 2 h, or 5 h.

[0289] Embodiment 164. The method of Embodiment 162 or 163, wherein, during the predetermined time period, the target molecule interacts with the subject to obtain a biological response that is indicative of the biological status of the subject or the portion of the subject.

[0290] Embodiment 165. The method of any one of Embodiments 156-164, wherein the target molecule is endogenous to the subject or the portion of the subject.

[0291] Embodiment 166. The method of any one of Embodiments 156-165, wherein the target molecule is exogenous to the subject or the portion of the subject.

[0292] Embodiment 167. The method of Embodiment 166, further comprising, prior to (b), administering the target molecule to the subject or the portion of the subject. [0293] Embodiment 168. The method of Embodiment 167, wherein the target molecule comprises partially or fully deuterated or carbon- 13 labeled fumarate, wherein the metabolic derivative of the target molecule comprises partially or fully deuterated or carbon- 13 labeled malate, and wherein the biological status of the subject or the portion of the subject comprises a presence or absence of necrosis in the subject or the portion of the subject.

[0294] Embodiment 169. The method of Embodiment 167, wherein the target molecule comprises partially or fully deuterated or carbon-13 labeled glucose, wherein the metabolic derivative of the target molecule comprises partially or fully deuterated or carbon- 13 labeled glutamine or partially or fully deuterated or carbon- 13 labeled glutamate, and wherein the biological status of the subject or the portion of the subject comprises an aerobic status of the subject or the portion of the subject or an anaerobic status of the subject or the portion of the subject.

[0295] Embodiment 170. The method of any one of Embodiments 156-169, wherein the target molecule comprises a first nuclear spin and a second nuclear spin, wherein the first nuclear spin and the second nuclear spin are of the same spin species, wherein a J-coupling between the first nuclear spin and the second nuclear spin is at least 1 hertz (Hz), and wherein:

[0296] Embodiment 171. The method of Embodiment 170, wherein the first nuclear spin and the second nuclear spin each comprise a proton nuclear spin, wherein the first nuclear spin and the second nuclear spin each comprise a carbon-13 nuclear spin or an isotopically enriched carbon- 13 nuclear spin, or wherein the first nuclear spin and the second nuclear spin each comprise a nitrogen-15 nuclear spin or an isotopically enriched carbon-13 nuclear spin.

[0297] Embodiment 172. The method of Embodiment 170 or 171, wherein the one or more pulse sequences preserve the target magnetization in a singlet state, suppress the non-target magnetization, and regenerate the target magnetization from the singlet state.

[0298] Embodiment 173. The method of any one of Embodiments 156-169, wherein the target molecule comprises a first nuclear spin and a second nuclear spin of a different spin species from the first nuclear spin, and wherein a J-coupling between the first nuclear spin and the second nuclear spin is at least 10 hertz (Hz).

[0299] Embodiment 174. The method of Embodiment 173, wherein the one or more pulse sequences preserve the target magnetization and suppress the non-target magnetization by transferring the target magnetization to magnetization on the second nuclear spin or by converting the target magnetization into a heteronuclear correlation between the first nuclear spin and the second nuclear spin, suppressing the non-target magnetization, and regenerating the target magnetization from the magnetization on the second nuclear spin or the heteronuclear correlation between the first nuclear spin and the second nuclear spin.

[0300] Embodiment 175. The method of Embodiment 173 or 174, wherein the one or more pulse sequences comprise one or more polarization transfer pulse sequences.

[0301] Embodiment 176. The method of any one of Embodiments 173-175, wherein the one or more pulse sequences comprise one or more insensitive nuclei enhanced by polarization transfer (INEPT) pulse sequences.

[0302] Embodiment 177. The method of Embodiment 173 or 174, wherein the one or more pulse sequences comprise one or more carbon- 13 -proton heteronuclear correlation pulse sequences.

[0303] Embodiment 178. The method of any one of Embodiments 156-177, wherein the one or more pulse sequences comprise one or more PulsePol pulse sequences. [0304] Embodiment 179. The method of any one of Embodiments 156-178, wherein the one or more pulse sequences comprise one or more gradient spoiling pulse sequences.

[0305] Embodiment 180. The method of any one of Embodiments 156-179, wherein the one or more pulse sequences comprise one or more radio-frequency (RF) spoiling pulse sequences.

[0306] Embodiment 181. The method of any one of Embodiments 156-180, wherein the one or more pulse sequences are configured to suppress the non-target magnetization in the other MRI- active molecules by a factor of at least 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, or 100,000.

[0307] Embodiment 182. A method comprising:

(a) identifying a target molecule in a subject or a portion of the subject, the target molecule comprising a first nuclear spin and a second nuclear spin;

(c) performing one or more pulse sequences to preserve the target magnetization in a singlet state, transfer the target magnetization to magnetization on the second nuclear spin, or convert the target magnetization into a heteronuclear correlation between the first nuclear spin and the second nuclear spin, suppress the non-target magnetization, and regenerate the target magnetization from the singlet state, the magnetization on the second nuclear spin, or the heteronuclear correlation utilizing a J-coupling network of the target molecule or the metabolic derivative of the target molecule;

[0308] Embodiment 183. The method of Embodiment 182, wherein the target molecule comprises at least one member selected from the group consisting of: a biologically active molecule, an antibody, a drug, an antibody-drug conjugate, a radionuclide, a radionuclide-drug conjugate, fumarate, glucose, and any carbon- 13 labeled, nitrogen- 15 labeled, partially deuterated, or fully deuterated analog thereof.

[0309] Embodiment 184. The method of Embodiment 182 or 183, wherein the metabolic derivative comprises at least one member selected from the group consisting of: a metabolic derivative of a biologically active molecule, a metabolic derivative of a drug, a metabolic derivative of an antibody-drug conjugate, a metabolic derivative of a radionuclide-drug conjugate, malate, glutamate, and any carbon- 13 labeled, nitrogen- 15 labeled, partially deuterated, or fully deuterated analog thereof.

[0310] Embodiment 185. The method of any one of Embodiments 182-184, wherein the nontarget magnetization comprises magnetization associated with water or fat in the subject or the portion of the subject.

[0311] Embodiment 186. The method of any one of Embodiments 182-185, wherein the biological status comprises at least one member selected from the group consisting of: a biological reaction of the subject or the portion of the subject to a biologically active molecule or a metabolic derivative of a biologically active molecule, a biological reaction of the subject or the portion of the subject to an antibody, a biological reaction of the subject or the portion of the subject to a drug or a metabolic derivative of a drug, a biological reaction of the subject or the portion of the subject to an antibody-drug conjugate or a metabolic derivative of an antibody-drug conjugate, a biological reaction of the subject or the portion of the subject to a radionuclide, a biological reaction of the subject or the portion of the subject to a radionuclidedrug conjugate or a metabolic derivative of a radionuclide-drug conjugate, a presence or absence of necrosis in the subject or the portion of the subject, an aerobic status of the subject or the portion of the subject, and an anaerobic status of the subject or the portion of the subject. [0312] Embodiment 187. The method of any one of claims 182-186, wherein the one or more pulse sequences are configured to reduce the non-target magnetization in the other MRI-active molecules by a factor of at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, or 100,000.

[0313] Embodiment 188. The method of any one of Embodiments 182-187, wherein (b) is performed a predetermined time period after (a).

[0314] Embodiment 189. The method of Embodiment 188, wherein the predetermined time period is at least 1 minute (min), 2 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 1 hour (h), 2 h, or 5 h.

[0315] Embodiment 190. The method of Embodiment 188 or 189, wherein, during the predetermined time period, the target molecule interacts with the subject to obtain a biological response that is indicative of the biological status of the subject or the portion of the subject.

[0316] Embodiment 191. The method of any one of Embodiments 182-190, wherein the target molecule is endogenous to the subject or the portion of the subject.

[0317] Embodiment 192. The method of any one of Embodiments 182-191, wherein the target molecule is exogenous to the subject or the portion of the subject.

[0318] Embodiment 193. The method of Embodiment 192, further comprising, prior to (b), administering the target molecule to the subject or the portion of the subject.

[0319] Embodiment 194. The method of Embodiment 193, wherein the target molecule comprises partially or fully deuterated or carbon- 13 labeled fumarate, wherein the metabolic derivative of the target molecule comprises partially or fully deuterated or carbon- 13 labeled malate, and wherein the biological status of the subject or the portion of the subject comprises a presence or absence of necrosis in the subject or the portion of the subject.

[0320] Embodiment 195. The method of Embodiment 193, wherein the target molecule comprises partially or fully deuterated or carbon-13 labeled glucose, wherein the metabolic derivative of the target molecule comprises partially or fully deuterated or carbon- 13 labeled glutamine or partially or fully deuterated or carbon- 13 labeled glutamate, and wherein the biological status of the subject or the portion of the subject comprises an aerobic status of the subject or the portion of the subject or an anaerobic status of the subject or the portion of the subject.

[0321] Embodiment 196. The method of any one of Embodiments 182-195, wherein the first nuclear spin and the second nuclear spin are of the same spin species, wherein a J-coupling between the first nuclear spin and the second nuclear spin is at least 1 hertz (Hz), and wherein:

[0322] Embodiment 197. The method of Embodiment 196, wherein the first nuclear spin and the second nuclear spin each comprise a proton nuclear spin, wherein the first nuclear spin and the second nuclear spin each comprise a carbon-13 nuclear spin or an isotopically enriched carbon- 13 nuclear spin, or wherein the first nuclear spin and the second nuclear spin each comprise a nitrogen-15 nuclear spin or an isotopically enriched carbon-13 nuclear spin.

[0323] Embodiment 198. The method of any one of Embodiments 182-195, wherein the second nuclear spin is of a different spin species from the first nuclear spin, and wherein a J-coupling between the first nuclear spin and the second nuclear spin is at least 10 hertz (Hz).

[0324] Embodiment 199. The method of Embodiment 198, wherein the one or more pulse sequences comprise one or more polarization transfer pulse sequences.

[0325] Embodiment 200. The method of Embodiment 198 or 199, wherein the one or more pulse sequences comprise one or more insensitive nuclei enhanced by polarization transfer (INEPT) pulse sequences.

[0326] Embodiment 201. The method of Embodiment 198, wherein the one or more pulse sequences comprise one or more carb on- 13 -proton heteronuclear correlation pulse sequences.

[0327] Embodiment 202. The method of any one of Embodiments 182-201, wherein the one or more pulse sequences comprise one or more PulsePol pulse sequences.

[0328] Embodiment 203. The method of any one of Embodiments 182-202, wherein the one or more pulse sequences comprise one or more gradient spoiling pulse sequences.

[0329] Embodiment 204. The method of any one of Embodiments 182-203, wherein the one or more pulse sequences comprise one or more radio-frequency (RF) spoiling pulse sequences.

[0330] Embodiment 205. A method comprising:

(a) providing carbon- 13 labeled fumarate to a subject or a portion of the subject;

(b) permitting at least a portion of carbon- 13 labeled fumarate to be converted to carbon- 13 labeled malate in the subject or the portion of the subject;

(c) subjecting the subject or the portion of the subject to a magnetic field in a magnetic resonance imaging (MRI) machine to induce a target magnetization in the carbon- 13 labeled fumarate and a derivative target magnetization in the carbon- 13 labeled malate and to induce a non-target magnetization in other MRI-active molecules in the subject or the portion of the subject;

(d) performing one or more first pulse sequences to preserve the target magnetization and to suppress the non-target magnetization and the derivative target magnetization by utilizing a J-coupling network of the carbon-13 labeled fumarate;

(e) obtaining one or more MRI images of the carbon- 13 labeled fumarate in the subject or the portion of the subject;

(f) performing one or more second pulse sequences to preserve the derivative target magnetization and to suppress the non-target magnetization and the target magnetization by utilizing a J-coupling network of the carbon- 13 labeled malate;

(g) obtaining one or more MRI images of the carbon- 13 labeled malate in the subject or the portion of the subject; and

(h) determining a biological status of the subject or the portion of the subject based on the one or more MRI images of the carbon- 13 labeled fumarate and the one or more MRI images of the carbon- 13 labeled malate.

[0331] Embodiment 206. The method of Embodiment 205, wherein the non-target magnetization comprises magnetization associated with water or fat in the subject or the portion of the subject.

[0332] Embodiment 207. The method of Embodiment 205 or 206, wherein the one or more first or second pulse sequences are configured to reduce the non-target magnetization in the other MRI-active molecules by a factor of at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, or 100,000.

[0333] Embodiment 208. The method of any one of Embodiments 205-207, wherein (c) is performed a predetermined time period after (a).

[0334] Embodiment 209. The method of Embodiment 208, wherein the predetermined time period is at least 1 minute (min), 2 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 1 hour (h), 2 h, or 5 h.

[0335] Embodiment 210. The method of any one of Embodiments 205-209, wherein the carbon- 13 labeled fumarate comprises a first nuclear spin and a second nuclear of the same spin species, wherein a J-coupling between of the first nuclear spin and the second nuclear spin is at least 1 Hz, and wherein:

(ii) the derivative molecule comprises a third nuclear spin, wherein a J-coupling between the third nuclear spin and the first nuclear spin is different than a J-coupling between the third nuclear spin and the second nuclear spin.

[0336] Embodiment 211. The method of Embodiment 210, wherein the first nuclear spin and the second nuclear spin each comprise a proton nuclear spin, wherein the first nuclear spin and the second nuclear spin each comprise a carbon-13 nuclear spin or an isotopically enriched carbon- 13 nuclear spin, or wherein the first nuclear spin and the second nuclear spin each comprise a nitrogen-15 nuclear spin or an isotopically enriched carbon-13 nuclear spin.

[0337] Embodiment 212. The method of Embodiment 210 or 211, wherein the one or more first pulse sequences preserve the target magnetization in a first singlet state, suppress the non-target magnetization and the derivative target magnetization, and regenerate the target magnetization from the first singlet state or wherein the one or more second pulse sequences preserve the derivative target magnetization in a second singlet state, suppress the non-target magnetization and the target magnetization, and regenerate the derivative target magnetization from the second singlet state.

[0338] Embodiment 213. The method of any one of Embodiments 205-209, wherein the carbon- 13 labeled fumarate comprises a first nuclear spin and a second nuclear spin of a different spin species from the first nuclear spin, and wherein a J-coupling between the first nuclear spin and the second nuclear spin is at least 10 hertz (Hz).

[0339] Embodiment 214. The method of Embodiment 213, wherein the one or more first or second pulse sequences comprise one or more polarization transfer pulse sequences.

[0340] Embodiment 215. The method of Embodiment 213 or 214, wherein the one or more first or second pulse sequences comprise one or more insensitive nuclei enhanced by polarization transfer (INEPT) pulse sequences.

[0341] Embodiment 216. The method of Embodiment 213, wherein the one or more pulse sequences comprise one or more carb on- 13 -proton heteronuclear correlation pulse sequences.

[0342] Embodiment 217. The method of any one of Embodiments 205-216, wherein the one or more first or second pulse sequences comprise one or more PulsePol pulse sequences.

[0343] Embodiment 218. The method of any one of Embodiments 205-217, wherein the one or more first or second pulse sequences comprise one or more gradient spoiling pulse sequences.

[0344] Embodiment 219. The method of any one of Embodiments 205-218, wherein the one or more first or second pulse sequences comprise one or more radio-frequency (RF) spoiling pulse sequences.

Claims

1. A method compri sing :

(a) identifying a target molecule in a subject or a portion of the subject:

(c) performing one or more pulse sequences to preserve the target magnetization and to suppress the non-target magnetization by a factor of at least 1,000 utilizing a J-coupling network of the target molecule or the metabolic derivative of the target molecule to selectively preserve the target magnetization;

2. The method of claim 1, wherein the target molecule comprises at least one member selected from the group consisting of: a biologically active molecule, an antibody, a drug, an antibody-drug conjugate, a radionuclide, a radionuclide-drug conjugate, fumarate, glucose, and any carbon- 13 labeled, nitrogen- 15 labeled, partially deuterated, or fully deuterated analog thereof.

3. The method of claim 1 or 2, wherein the metabolic derivative comprises at least one member selected from the group consisting of: a metabolic derivative of a biologically active molecule, a metabolic derivative of a drug, a metabolic derivative of an antibodydrug conjugate, a metabolic derivative of a radionuclide-drug conjugate, malate, glutamate, and any carbon- 13 labeled, nitrogen- 15 labeled, partially deuterated, or fully deuterated analog thereof.

4. The method of any one of claims 1-3, wherein the non-target magnetization comprises magnetization associated with water or fat in the subject or the portion of the subject.

5. The method of any one of claims 1-4, wherein the biological status comprises at least one member selected from the group consisting of: a biological reaction of the subject or the portion of the subject to a biologically active molecule or a metabolic derivative of a biologically active molecule, a biological reaction of the subject or the portion of the subject to an antibody, a biological reaction of the subject or the portion of the subject to a drug or a metabolic derivative of a drug, a biological reaction of the subject or the portion of the subject to an antibody-drug conjugate or a metabolic derivative of an antibody-drug conjugate, a biological reaction of the subject or the portion of the subject to a radionuclide, a biological reaction of the subject or the portion of the subject to a radionuclide-drug conjugate or a metabolic derivative of a radionuclide-drug conjugate, a presence or absence of necrosis in the subject or the portion of the subject, an aerobic status of the subject or the portion of the subject, and an anaerobic status of the subject or the portion of the subject.

6. The method of any one of claims 1-5, wherein the one or more pulse sequences are configured to reduce the non-target magnetization in the other MRI-active molecules by a factor of at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, or 100,000.

7. The method of any one of claims 1-6, wherein (b) is performed a predetermined time period after (a).

8. The method of claim 7, wherein the predetermined time period is at least 1 minute (min),

2 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min,

55 min, 1 hour (h), 2 h, or 5 h.

9. The method of claim 7 or 8, wherein, during the predetermined time period, the target molecule interacts with the subject to obtain a biological response that is indicative of the biological status of the subject or the portion of the subject.

10. The method of any one of claims 1-9, wherein the target molecule is endogenous to the subject or the portion of the subject.

11. The method of any one of claims 1-10, wherein the target molecule is exogenous to the subject or the portion of the subject.

12. The method of claim 11, further comprising, prior to (b), administering the target molecule to the subject or the portion of the subject.

13. The method of claim 12, wherein the target molecule comprises partially or fully deuterated or carbon- 13 labeled fumarate, wherein the metabolic derivative of the target molecule comprises partially or fully deuterated or carbon- 13 labeled malate, and wherein the biological status of the subject or the portion of the subject comprises a presence or absence of necrosis in the subject or the portion of the subject.

14. The method of claim 13, wherein the target molecule comprises partially or fully deuterated or carbon- 13 labeled glucose, wherein the metabolic derivative of the target molecule comprises partially or fully deuterated or carbon- 13 labeled glutamine or partially or fully deuterated or carbon- 13 labeled glutamate, and wherein the biological status of the subject or the portion of the subject comprises an aerobic status of the subject or the portion of the subject or an anaerobic status of the subject or the portion of the subject.

15. The method of any one of claims 1-14, wherein the target molecule comprises a first nuclear spin and a second nuclear spin, wherein the first nuclear spin and the second nuclear spin are of the same spin species, wherein a J-coupling between the first nuclear spin and the second nuclear spin is at least 1 hertz (Hz), and wherein:

(ii) the target molecule further comprises a third nuclear spin, wherein a J- coupling between the third nuclear spin and the first nuclear spin is different than a J-coupling between the third nuclear spin and the second nuclear spin.

16. The method of claim 15, wherein the first nuclear spin and the second nuclear spin each comprise a proton nuclear spin, wherein the first nuclear spin and the second nuclear spin each comprise a carbon-13 nuclear spin or an isotopically enriched carbon-13 nuclear spin, or wherein the first nuclear spin and the second nuclear spin each comprise a nitrogen-15 nuclear spin or an isotopically enriched carbon-13 nuclear spin.

17. The method of claim 15 or 16, wherein the one or more pulse sequences preserve the target magnetization in a singlet state, suppress the non-target magnetization, and regenerate the target magnetization from the singlet state.

18. The method of any one of claims 1-14, wherein the target molecule comprises a first nuclear spin and a second nuclear spin of a different spin species from the first nuclear spin, and wherein a J-coupling between the first nuclear spin and the second nuclear spin is at least 10 hertz (Hz).

19. The method of claim 18, wherein the one or more pulse sequences preserve the target magnetization and suppress the non-target magnetization by transferring the target magnetization to magnetization on the second nuclear spin or by converting the target magnetization into a heteronuclear correlation between the first nuclear spin and the second nuclear spin, suppressing the non-target magnetization, and regenerating the target magnetization from the magnetization on the second nuclear spin or the heteronuclear correlation between the first nuclear spin and the second nuclear spin.

20. The method of claim 18 or 19, wherein the one or more pulse sequences comprise one or more polarization transfer pulse sequences.

21. The method of any one of claims 18-20, wherein the one or more pulse sequences comprise one or more insensitive nuclei enhanced by polarization transfer (INEPT) pulse sequences.

22. The method of claim 18 or 19, wherein the one or more pulse sequences comprise one or more carbon- 13 -proton heteronuclear correlation pulse sequences

23. The method of any one of claims 1-22, wherein the one or more pulse sequences comprise one or more PulsePol pulse sequences.

24. The method of any one of claims 1-23, wherein the one or more pulse sequences comprise one or more gradient spoiling pulse sequences.

25. The method of any one of claims 1-24, wherein the one or more pulse sequences comprise one or more radio-frequency (RF) spoiling pulse sequences.

26. The method of any one of claims 1-25, wherein the one or more pulse sequences are configured to suppress the non-target magnetization in the other MRI-active molecules by a factor of at least 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000,

20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, or 100,000.

27. A method comprising:

(a) identifying a target molecule in a subj ect or a portion of the subj ect, the target molecule comprising a first nuclear spin and a second nuclear spin;

28. The method of claim 27, wherein the target molecule comprises at least one member selected from the group consisting of: a biologically active molecule, an antibody, a drug, an antibody-drug conjugate, a radionuclide, a radionuclide-drug conjugate, fumarate, glucose, and any carbon- 13 labeled, nitrogen- 15 labeled, partially deuterated, or fully deuterated analog thereof.

29. The method of claim 27 or 28, wherein the metabolic derivative comprises at least one member selected from the group consisting of: a metabolic derivative of a biologically active molecule, a metabolic derivative of a drug, a metabolic derivative of an antibodydrug conjugate, a metabolic derivative of a radionuclide-drug conjugate, malate, glutamate, and any carbon- 13 labeled, nitrogen- 15 labeled, partially deuterated, or fully deuterated analog thereof.

30. The method of any one of claims 27-29, wherein the non-target magnetization comprises magnetization associated with water or fat in the subject or the portion of the subject.

31. The method of any one of claims 27-30, wherein the biological status comprises at least one member selected from the group consisting of: a biological reaction of the subject or the portion of the subject to a biologically active molecule or a metabolic derivative of a biologically active molecule, a biological reaction of the subject or the portion of the subject to an antibody, a biological reaction of the subject or the portion of the subject to a drug or a metabolic derivative of a drug, a biological reaction of the subject or the portion of the subject to an antibody-drug conjugate or a metabolic derivative of an antibody-drug conjugate, a biological reaction of the subject or the portion of the subject to a radionuclide, a biological reaction of the subject or the portion of the subject to a radionuclide-drug conjugate or a metabolic derivative of a radionuclide-drug conjugate, a presence or absence of necrosis in the subject or the portion of the subject, an aerobic status of the subject or the portion of the subject, and an anaerobic status of the subject or the portion of the subject.

32. The method of any one of claims 27-31, wherein the one or more pulse sequences are configured to reduce the non-target magnetization in the other MRI-active molecules by a factor of at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, or 100,000.

33. The method of any one of claims 27-32, wherein (b) is performed a predetermined time period after (a).

34. The method of claim 33, wherein the predetermined time period is at least 1 minute (min), 2 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 1 hour (h), 2 h, or 5 h.

35. The method of claim 33 or 34, wherein, during the predetermined time period, the target molecule interacts with the subject to obtain a biological response that is indicative of the biological status of the subject or the portion of the subject.

36. The method of any one of claims 27-35, wherein the target molecule is endogenous to the subject or the portion of the subject.

37. The method of any one of claims 27-36, wherein the target molecule is exogenous to the subject or the portion of the subject.

38. The method of claim 37, further comprising, prior to (b), administering the target molecule to the subject or the portion of the subject.

39. The method of claim 38, wherein the target molecule comprises partially or fully deuterated or carbon- 13 labeled fumarate, wherein the metabolic derivative of the target molecule comprises partially or fully deuterated or carbon- 13 labeled malate, and wherein the biological status of the subject or the portion of the subject comprises a presence or absence of necrosis in the subject or the portion of the subject.

40. The method of claim 38, wherein the target molecule comprises partially or fully deuterated or carbon- 13 labeled glucose, wherein the metabolic derivative of the target molecule comprises partially or fully deuterated or carbon- 13 labeled glutamine or partially or fully deuterated or carbon- 13 labeled glutamate, and wherein the biological status of the subject or the portion of the subject comprises an aerobic status of the subject or the portion of the subject or an anaerobic status of the subject or the portion of the subject.

41. The method of any one of claims 27-40, wherein the first nuclear spin and the second nuclear spin are of the same spin species, wherein a J-coupling between the first nuclear spin and the second nuclear spin is at least 1 hertz (Hz), and wherein:

42. The method of claim 41, wherein the first nuclear spin and the second nuclear spin each comprise a proton nuclear spin, wherein the first nuclear spin and the second nuclear spin each comprise a carbon-13 nuclear spin or an isotopically enriched carbon-13 nuclear spin, or wherein the first nuclear spin and the second nuclear spin each comprise a nitrogen-15 nuclear spin or an isotopically enriched carbon-13 nuclear spin.

43. The method of any one of claims 27-40, wherein the second nuclear spin is of a different spin species from the first nuclear spin, and wherein a J-coupling between the first nuclear spin and the second nuclear spin is at least 10 hertz (Hz).

44. The method of claim 43, wherein the one or more pulse sequences comprise one or more polarization transfer pulse sequences.

45. The method of claim 43 or 44, wherein the one or more pulse sequences comprise one or more insensitive nuclei enhanced by polarization transfer (INEPT) pulse sequences.

46. The method of claim 43, wherein the one or more pulse sequences comprise one or more carb on- 13 -proton heteronuclear correlation pulse sequences.

47. The method of any one of claims 27-46, wherein the one or more pulse sequences comprise one or more PulsePol pulse sequences.

48. The method of any one of claims 27-47, wherein the one or more pulse sequences comprise one or more gradient spoiling pulse sequences.

49. The method of any one of claims 27-48, wherein the one or more pulse sequences comprise one or more radio-frequency (RF) spoiling pulse sequences.

50. A method comprising:

(i) providing carbon- 13 labeled fumarate to a subject or a portion of the subject;

(ii) permitting at least a portion of carbon- 13 labeled fumarate to be converted to carbon- 13 labeled malate in the subject or the portion of the subject;

(iii)subjecting the subject or the portion of the subject to a magnetic field in a magnetic resonance imaging (MRI) machine to induce a target magnetization in the carbon- 13 labeled fumarate and a derivative target magnetization in the carbon- 13 labeled malate and to induce a non-target magnetization in other MRI-active molecules in the subject or the portion of the subject;

(iv)performing one or more first pulse sequences to preserve the target magnetization and to suppress the non-target magnetization and the derivative target magnetization by utilizing a J-coupling network of the carbon- 13 labeled fumarate;

(v) obtaining one or more MRI images of the carbon- 13 labeled fumarate in the subject or the portion of the subject;

(vi)performing one or more second pulse sequences to preserve the derivative target magnetization and to suppress the non-target magnetization and the target magnetization by utilizing a J-coupling network of the carbon- 13 labeled malate;

(vii) obtaining one or more MRI images of the carbon- 13 labeled malate in the subject or the portion of the subject; and

(viii) determining a biological status of the subj ect or the portion of the subj ect based on the one or more MRI images of the carbon- 13 labeled fumarate and the one or more MRI images of the carbon- 13 labeled malate.

51. The method of claim 50, wherein the non-target magnetization comprises magnetization associated with water or fat in the subject or the portion of the subject.

52. The method of claim 50 or 51, wherein the one or more first or second pulse sequences are configured to reduce the non-target magnetization in the other MRI-active molecules by a factor of at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, or 100,000.

53. The method of any one of claims 50-52, wherein (c) is performed a predetermined time period after (a).

54. The method of claim 53, wherein the predetermined time period is at least 1 minute (min), 2 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 1 hour (h), 2 h, or 5 h.

55. The method of any one of claims 50-54, wherein the carbon-13 labeled fumarate comprises a first nuclear spin and a second nuclear of the same spin species, wherein a J-coupling between of the first nuclear spin and the second nuclear spin is at least 1 Hz, and wherein:

(ii) the derivative molecule comprises a third nuclear spin, wherein a J- coupling between the third nuclear spin and the first nuclear spin is different than a J-coupling between the third nuclear spin and the second nuclear spin.

56. The method of claim 55, wherein the first nuclear spin and the second nuclear spin each comprise a proton nuclear spin, wherein the first nuclear spin and the second nuclear spin each comprise a carbon-13 nuclear spin or an isotopically enriched carbon-13 nuclear spin, or wherein the first nuclear spin and the second nuclear spin each comprise a nitrogen-15 nuclear spin or an isotopically enriched carbon-13 nuclear spin

57. The method of claim 55 or 56, wherein the one or more first pulse sequences preserve the target magnetization in a first singlet state, suppress the non-target magnetization and the derivative target magnetization, and regenerate the target magnetization from the first singlet state or wherein the one or more second pulse sequences preserve the derivative target magnetization in a second singlet state, suppress the non-target magnetization and the target magnetization, and regenerate the derivative target magnetization from the second singlet state.

58. The method of any one of claims 50-54, wherein the carbon-13 labeled fumarate comprises a first nuclear spin and a second nuclear spin of a different spin species from the first nuclear spin, and wherein a J-coupling between the first nuclear spin and the second nuclear spin is at least 10 hertz (Hz).

59. The method of claim 58, wherein the one or more first or second pulse sequences comprise one or more polarization transfer pulse sequences.

60. The method of claim 58 or 59, wherein the one or more first or second pulse sequences comprise one or more insensitive nuclei enhanced by polarization transfer (INEPT) pulse sequences.

61. The method of claim 58, wherein the one or more pulse sequences comprise one or more carb on- 13 -proton heteronuclear correlation pulse sequences.

62. The method of any one of claims 50-61, wherein the one or more first or second pulse sequences comprise one or more PulsePol pulse sequences.

63. The method of any one of claims 50-62, wherein the one or more first or second pulse sequences comprise one or more gradient spoiling pulse sequences.

64. The method of any one of claims 50-63, wherein the one or more first or second pulse sequences comprise one or more radio-frequency (RF) spoiling pulse sequences.