Multi-axis fields boost SABRE hyperpolarization.

The inherently low signal-to-noise ratio of NMR and MRI is now being addressed by hyperpolarization methods. For example, iridium-based catalysts that reversibly bind both parahydrogen and ligands in solution can hyperpolarize protons (SABRE) or heteronuclei (X-SABRE) on a wide variety of ligands, using a complex interplay of spin dynamics and chemical exchange processes, with common signal enhancements between 103 and 104. This does not approach obvious theoretical limits, and further enhancement would be valuable in many applications (such as imaging mM concentration species in vivo). Most SABRE/X-SABRE implementations require far lower fields (µT-mT) than standard magnetic resonance (>1T), and this gives an additional degree of freedom: the ability to fully modulate fields in three dimensions. However, this has been underexplored because the standard simplifying theoretical assumptions in magnetic resonance need to be revisited. Here, we take a different approach, an evolutionary strategy algorithm for numerical optimization, multi-axis computer-aided heteronuclear transfer enhancement for SABRE (MACHETE-SABRE). We find nonintuitive but highly efficient multiaxial pulse sequences which experimentally can produce a sevenfold improvement in polarization over continuous excitation. This approach optimizes polarization differently than traditional methods, thus gaining extra efficiency.

Contrast mechanisms in pump-probe microscopy of melanin.

Pump-probe microscopy of melanin in tumors has been proposed to improve diagnosis of malignant melanoma, based on the hypothesis that aggressive cancers disaggregate melanin structure. However, measured signals of melanin are complex superpositions of multiple nonlinear processes, which makes interpretation challenging. Polarization control during measurement and data fitting are used to decompose signals of melanin into their underlying molecular mechanisms. We then identify the molecular mechanisms that are most susceptible to melanin disaggregation and derive false-coloring schemes to highlight these processes in biological tissue. We demonstrate that false-colored images of a small set of melanoma tumors correlate with clinical concern. More generally, our systematic approach of decomposing pump-probe signals can be applied to a multitude of different samples.

Design, synthesis and evaluation of 15N- and 13C-labeled molecular probes as hyperpolarized nitric oxide sensors.

Nitric oxide (NO) is an important signaling molecule involved in a wide range of biological processes. Development of non-invasive, real-time detection of NO is greatly desired yet remains challenging. Here we report the design and development of novel 15N- and 13C-labeled NO-sensing probes for hyperpolarized nuclear magnetic resonance (HP-NMR) studies. These probes undergo selective and rapid reaction with NO to generate in situ AZO-products that can be monitored with distinguishable NMR signals as a read-out. This study also allows for a direct comparison of the 15N and 13C nuclei performances in hyperpolarized reaction-based probes. The simple and general SABRE-SHEATH hyperpolarization method works on the 15N- and 13C-NO-sensing probes. Measured long spin-lattice relaxation (T1) values, especially for 15N-NO probes, will allow for real-time reaction-based imaging of NO.

SABRE enhancement with oscillating pulse sequences.

SABRE (Signal Amplification by Reversible Exchange) methods provide a simple, fast, and cost-effective method to hyperpolarize a wide variety of molecules in solution, and have been demonstrated with protons and, more recently, with heteronuclei (X-SABRE). Here, we present several oscillating pulse sequences that use magnetic fields far away from the resonance condition of continuous excitation and can commonly triple the polarization. An analysis with average Hamiltonian theory indicates that the oscillating pulse, in effect, adjusts the J-couplings between hydrides and target nuclei and that a much weaker coupling produces maximum polarization. This theoretical treatment, combined with simulations and experiment, shows substantial magnetization improvements relative to traditional X-SABRE methods. It also shows that, in contrast to most pulse sequence applications, waveforms with reduced time symmetry in the toggling frame make magnetization generation more robust to experimental imperfections.

Phase coherent excitation of SABRE permits simultaneous hyperpolarization of multiple targets at high magnetic field.

Hyperpolarization methods in magnetic resonance overcome sensitivity limitations, especially for low-γ nuclei such as 13C and 15N. Signal Amplification By Reversible Exchange (SABRE) and extended SABRE (X-SABRE) are efficient and low-cost methods for generating large polarizations on a variety of nuclei, but they most commonly use low magnetic fields (µT-mT). High field approaches, where hyperpolarization is generated directly in the spectrometer, are potentially much more convenient but have been limited to selectively hyperpolarize single targets. Here we introduce a new pulse sequence-based approach that affords broadband excitation of SABRE hyperpolarization at high magnetic fields without having to tailor pulse sequence parameters to specific targets. This permits simultaneous hyperpolarization of multiple targets for the first time at high field and offers a direct approach to integration of high-field SABRE hyperpolarization into routine NMR applications, such as NMR-based metabonomics and biomolecular NMR.

Interplay of Near-Zero-Field Dephasing, Rephasing, and Relaxation Dynamics and [1-13C]Pyruvate Polarization Transfer Efficiency in Pulsed SABRE-SHEATH.

Hyperpolarized [1-13C]pyruvate is a revolutionary molecular probe enabling ultrafast metabolic MRI scans in 1 min. This technology is now under evaluation in over 30 clinical trials, which employ dissolution Dynamic Nuclear Polarization (d-DNP) to prepare a batch of the contrast agent; however, d-DNP technology is slow and expensive. The emerging SABRE-SHEATH hyperpolarization technique enables fast (under 1 min) and robust production of hyperpolarized [1-13C]pyruvate via simultaneous chemical exchange of parahydrogen and pyruvate on IrIMes hexacoordinate complexes. Here, we study the application of microtesla pulses to investigate their effect on C-13 polarization efficiency, compared to that of conventional SABRE-SHEATH employing a static field (∼0.4 µT), to provide the matching conditions of polarization transfer from parahydrogen-derived hydrides to the 13C-1 nucleus. Our results demonstrate that using square-microtesla pulses with optimized parameters can produce 13C-1 polarization levels of up to 14.8% (when detected, averaging over all resonances), corresponding to signal enhancement by over 122,000-fold at the clinically relevant field of 1.4 T. We anticipate that our results can be directly translated to other structurally similar biomolecules such as [1-13C]α-ketoglutarate and [1-13C]α-ketoisocaproate. Moreover, other more advanced pulse shapes can potentially further boost heteronuclear polarization attainable via pulsed SABRE-SHEATH.

Beyond intensity modulation: new approaches to pump-probe microscopy.

Pump-probe microscopy is an emerging nonlinear imaging technique based on high repetition rate lasers and fast intensity modulation. Here, we present new methods for pump-probe microscopy that keep the beam intensity constant and instead modulate the inter-pulse time delay or the relative polarization. These techniques can improve image quality for samples that have poor heat dissipation or long-lived radiative states and can selectively address nonlinear interactions in the sample. We experimentally demonstrate this approach and point out the advantages over conventional intensity modulation.

Application of 15N2-Diazirines as a Versatile Platform for Hyperpolarization of Biological Molecules by d-DNP.

15N2-Diazirines represent an attractive class of imaging tags for hyperpolarized magnetic resonance imaging (HP-MRI), offering desirable biocompatibility, ease of incorporation into a variety of molecules, and ability to deliver long-lasting polarization. We have recently established hyperpolarization of 15N2-diazirines in organic solvents using SABRE-Shield Enables Alignment Transfer to Heteronuclei (SABRE-SHEATH). Yet, the current challenge of SABRE-SHEATH in water, specifically poor polarization efficiency, presents a barrier in examining the practical use of 15N2-diazirines for HP-MRI. Herein, we show that efficient polarization of diverse 15N2-diazirine-labeled molecules in water can be readily achieved by dissolution dynamic nuclear polarization (d-DNP), a hyperpolarization technique used in clinical practice. Hyperpolarization by d-DNP also demonstrates greater enhancement for long-lasting 15N signals, in comparison with SABRE-SHEATH. Various biologically important molecules are studied in this work, including amino acid, sugar, and drug compounds, demonstrating the great potential of 15N2-diazirines as molecular tags in broad biomedical and clinical applications.

Crossed-beam pump-probe microscopy.

We present a new imaging method for pump-probe microscopy that explores non-collinear excitation. This method (crossed-beam pump-probe microscopy, or CBPM) can significantly improve the axial resolution when imaging through low-NA lenses, providing an alternative way for depth-resolved, large field-of-view imaging. We performed a proof-of-concept demonstration, characterized CBPM's resolution using different imaging lenses, and measured an enhanced axial resolution for certain types of low-NA lenses.

"Direct" 13 C Hyperpolarization of 13 C-Acetate by MicroTesla NMR Signal Amplification by Reversible Exchange (SABRE).

Herein, we demonstrate "direct" 13 C hyperpolarization of 13 C-acetate via signal amplification by reversible exchange (SABRE). The standard SABRE homogeneous catalyst [Ir-IMes; [IrCl(COD)(IMes)], (IMes=1,3-bis(2,4,6-trimethylphenyl), imidazole-2-ylidene; COD=cyclooctadiene)] was first activated in the presence of an auxiliary substrate (pyridine) in alcohol. Following addition of sodium 1-13 C-acetate, parahydrogen bubbling within a microtesla magnetic field (i.e. under conditions of SABRE in shield enables alignment transfer to heteronuclei, SABRE-SHEATH) resulted in positive enhancements of up to ≈100-fold in the 13 C NMR signal compared to thermal equilibrium at 9.4 T. The present results are consistent with a mechanism of "direct" transfer of spin order from parahydrogen to 13 C spins of acetate weakly bound to the catalyst, under conditions of fast exchange with respect to the 13 C acetate resonance, but we find that relaxation dynamics at microtesla fields alter the optimal matching from the traditional SABRE-SHEATH picture. Further development of this approach could lead to new ways to rapidly, cheaply, and simply hyperpolarize a broad range of substrates (e.g. metabolites with carboxyl groups) for various applications, including biomedical NMR and MRI of cellular and in vivo metabolism.

Iodonitrene in Action: Direct Transformation of Amino Acids into Terminal Diazirines and 15N2-Diazirines and Their Application as Hyperpolarized Markers.

A one-pot metal-free conversion of unprotected amino acids to terminal diazirines has been developed using phenyliodonium diacetate (PIDA) and ammonia. This PIDA-mediated transformation occurs via three consecutive reactions and involves an iodonitrene intermediate. This method is tolerant to most functional groups found on the lateral chain of amino acids, it is operationally simple, and it can be scaled up to provide multigram quantities of diazirine. Interestingly, we also demonstrated that this transformation could be applied to dipeptides without racemization. Furthermore, 14N2 and 15N2 isotopomers can be obtained, emphasizing a key trans-imination step when using 15NH3. In addition, we report the first experimental observation of 14N/15N isotopomers directly creating an asymmetric carbon. Finally, the 15N2-diazirine from l-tyrosine was hyperpolarized by a parahydrogen-based method (SABRE-SHEATH), demonstrating the products' utility as hyperpolarized molecular tag.

Hyperpolarizing Concentrated Metronidazole 15 NO2 Group over Six Chemical Bonds with More than 15 % Polarization and a 20†Minute Lifetime.

The NMR hyperpolarization of uniformly 15 N-labeled [15 N3 ]metronidazole is demonstrated by using SABRE-SHEATH. In this antibiotic, the 15 NO2 group is hyperpolarized through spin relays created by 15 N spins in [15 N3 ]metronidazole, and the polarization is transferred from parahydrogen-derived hydrides over six chemical bonds. In less than a minute of parahydrogen bubbling at approximately 0.4 µT, a high level of nuclear spin polarization (P15N ) of around 16 % is achieved on all three 15 N sites. This product of 15 N polarization and concentration of 15 N spins is around six-fold better than any previous value determined for 15 N SABRE-derived hyperpolarization. At 1.4 T, the hyperpolarized state persists for tens of minutes (relaxation time, T1 ≈10 min). A novel synthesis of uniformly 15 N-enriched metronidazole is reported with a yield of 15 %. This approach can potentially be used for synthesis of a wide variety of in vivo metabolic probes with potential uses ranging from hypoxia sensing to theranostic imaging.

Visualizing the impact of chloride addition on the microscopic carrier dynamics of MAPbI3 thin films using femtosecond transient absorption microscopy.

The spatial heterogeneity of carrier dynamics in mixed halide perovskite CH3NH3PbI3-xClx thin films with a range of different chloride additions is mapped using femtosecond transient absorption microscopy (TAM). The comparison of TAM images of fibrous and granular polycrystalline CH3NH3PbI3-xClx films indicates that the impact of chloride addition on the local heterogeneity of carrier dynamics is highly dependent on the film preparation method and the resulting morphology. In addition to signals of pristine CH3NH3PbI3, CH3NH3PbI3-xClx films with a fibrous structure show long-lived excited state absorption (ESA) signals in localized, microscopic regions. The ESA signal exhibits transient absorption with a rise time of about 5 ps after the excitation pulse, indicating that these distinct micrograins have preferential carrier trapping properties. The chemical composition of these micrograins does not differ detectably from their surroundings. In contrast, in CH3NH3PbI3-xClx films with a granular structure, Cl addition does not seem to affect the charge carrier dynamics. These results provide insight into the localized effects of halide mixing and on the resulting photophysical properties of mixed halide perovskite materials on the micrometer length scale.

Selective hyperpolarization of heteronuclear singlet states via pulsed microtesla SABRE.

Signal Amplification By Reversible Exchange (SABRE) and its heteronuclear variant SABRE in SHield Enables Alignment Transfer to Heteronuclei create large nuclear magnetization in target ligands, exploiting level crossings in an iridium catalyst that transiently binds both the ligands and parahydrogen. This requires a specific, small magnetic field to match Zeeman splittings to scalar couplings. Here, we explore a different strategy, direct creation of heteronuclear singlet states in the target ligands, which produces enhanced signals at other field strengths, including zero field. We also show that pulsed methods (including pulsed field nulling) coherently and selectively pump such singlets, affording a significant enhancement on the resulting hyperpolarization.

Terminal Diazirines Enable Reverse Polarization Transfer from 15 N2 Singlets.

Diazirine moieties are chemically stable and have been incorporated into biomolecules without impediment of biological activity. The 15 N2 labeled diazirines are appealing motifs for hyperpolarization supporting relaxation protected states with long-lived lifetimes. The (-CH15 N2 ) diazirine groups investigated here are analogues to methyl groups, which provides the opportunity to transfer polarization stored on a relaxation protected (-CH15 N2 ) moiety to 1 H, thus combining the advantages of long lifetimes of 15 N polarization with superior sensitivity of 1 H detection. Despite the proximity of 1 H to 15 N nuclei in the diazirine moiety, 15 N T1 times of up to (4.6±0.4) min and singlet lifetimes Ts of up to (17.5±3.8) min are observed. Furthermore, we found terminal diazirines to support hyperpolarized 1 H2 singlet states in CH2 groups of chiral molecules. The singlet lifetime of 1 H singlets is up to (9.2±1.8) min, thus exceeding 1 H T1 relaxation time (at 8.45 T) by a factor of ≈100.

Spectroscopic Differentiation and Microscopic Imaging of Red Organic Pigments Using Optical Pump-Probe Contrast.

Analysis of red organic pigments in artworks (and in forensics applications) is challenging, because conventional nondestructive mapping techniques provide little contrast, and most chemical analyses with high specificity require sample removal. Here we demonstrate a new optical approach, pump-probe microscopy, for the analysis of red organic pigments. We investigate Carmine naccarat, Lac dye, purpurin, alizarin, madder lake, and eosin Y and show that their intrinsic photophysical properties produce distinctive pump-probe spectra. We utilize this contrast for high-resolution, three-dimensional imaging without the need for physical sample removal. Lastly, we highlight the potential of pump-probe microscopy as an analytical tool for forensics of other types of organic colorants by investigating a series of automotive paints.

Direct Hyperpolarization of Nitrogen-15 in Aqueous Media with Parahydrogen in Reversible Exchange.

Signal amplification by reversible exchange (SABRE) is an inexpensive, fast, and even continuous hyperpolarization technique that uses para-hydrogen as hyperpolarization source. However, current SABRE faces a number of stumbling blocks for translation to biochemical and clinical settings. Difficulties include inefficient polarization in water, relatively short-lived 1H-polarization, and relatively limited substrate scope. Here we use a water-soluble polarization transfer catalyst to hyperpolarize nitrogen-15 in a variety of molecules with SABRE-SHEATH (SABRE in shield enables alignment transfer to heteronuclei). This strategy works in pure H2O or D2O solutions, on substrates that could not be hyperpolarized in traditional 1H-SABRE experiments, and we record 15N T1 relaxation times of up to 2 min.

Toward Hyperpolarized 19 F Molecular Imaging via Reversible Exchange with Parahydrogen.

Fluorine-19 has high NMR detection sensitivity-similar to that of protons-owing to its large gyromagnetic ratio and high natural abundance (100 %). Unlike protons, however, fluorine-19 (19 F) has a negligible occurrence in biological objects, as well as a more sensitive chemical shift. As a result, in vivo 19 F NMR spectroscopy and MR imaging offer advantages of negligible background signal and sensitive reporting of the local molecular environment. Here we report on NMR hyperpolarization of 19 F nuclei using reversible exchange reactions with parahydrogen gas as the source of nuclear spin order. NMR signals of 3-fluoropyridine were enhanced by ≈100 fold, corresponding to 0.3 % 19 F nuclear spin polarization (at 9.4 T), using about 50 % parahydrogen. While future optimization efforts will likely significantly increase the hyperpolarization levels, we already demonstrate the utility of 19 F hyperpolarization for high-resolution hyperpolarized 19 F imaging and hyperpolarized 19 F pH sensing.

The Absence of Quadrupolar Nuclei Facilitates Efficient 13 C Hyperpolarization via Reversible Exchange with Parahydrogen.

Nuclear spin hyperpolarization techniques are revolutionizing the field of 13 C molecular MRI. While dissolution dynamic nuclear polarization (d-DNP) is currently the leading technique, it is generally slow (requiring ≈1 h) and costly (≈$USD106 ). As a consequence of carbon's central place in biochemistry, tremendous progress using 13 C d-DNP bioimaging has been demonstrated to date including a number of clinical trials. Despite numerous attempts to develop alternatives to d-DNP, the competing methods have faced significant translational challenges. Efficient hyperpolarization of 15 N, 31 P, and other heteronuclei using signal amplification by reversible exchange (SABRE) has been reported in 2015, but extension of this technique to 13 C has proven to be challenging. Here, we present efficient hyperpolarization of 13 C nuclei using micro-Tesla SABRE. Up to ca. 6700-fold enhancement of nuclear spin polarization at 8.45 T is achieved within seconds, corresponding to P13C ≈4.4 % using 50 % parahydrogen (P13C >14 % would be feasible using more potent ≈100 % parahydrogen). Importantly, the 13 C polarization achieved via SABRE strongly depends not only upon spin-lattice relaxation, but also upon the presence of 15 N (I=1/2) versus quadrupolar 14 N (I=1) spins in the site binding the hexacoordinate Ir atom of the catalytic complex. We show that different 13 C nuclei in the test molecular frameworks-pyridine and acetonitrile-can be hyperpolarized, including 13 C sites up to five chemical bonds away from the exchangeable hydrides. The presented approach is highly scalable and can be applied to a rapidly growing number of biomolecules amendable to micro-Tesla SABRE.