Direct and cost-efficient hyperpolarization of long-lived nuclear spin states on universal (15)N2-diazirine molecular tags.

Conventional magnetic resonance (MR) faces serious sensitivity limitations which can be overcome by hyperpolarization methods, but the most common method (dynamic nuclear polarization) is complex and expensive, and applications are limited by short spin lifetimes (typically seconds) of biologically relevant molecules. We use a recently developed method, SABRE-SHEATH, to directly hyperpolarize (15)N2 magnetization and long-lived (15)N2 singlet spin order, with signal decay time constants of 5.8 and 23 minutes, respectively. We find >10,000-fold enhancements generating detectable nuclear MR signals that last for over an hour. (15)N2-diazirines represent a class of particularly promising and versatile molecular tags, and can be incorporated into a wide range of biomolecules without significantly altering molecular function.

Accessing long lived (1)H states via (2)H couplings.

In this paper we demonstrate long-lived states involving a pair of chemically equivalent protons, with lifetimes ∼30 times T1 up to a total lifetime of ∼117s at high field (8.45T). This is demonstrated on trans-ethylene-d2 in solution, where magnetic inequivalence gives access to the long-lived states. It is shown that the remaining J-coupling between the two quadrupolar deuterium spins, JQQ, splits the conditions for optimally generating proton singlet states. Detailed simulations of the spin evolution are performed, shedding light on the coherent evolution during singlet-triplet conversion as well as on the incoherent evolution that causes relaxation. Subsequently, the simulations are compared with experimental results validating the theoretical insights. Possible applications include storage of hyperpolarization in the proton long-lived state. Of particular interest may be utilization of parahydrogen induced polarization to directly induce the examined long-lived states.

Long-lived polarization protected by symmetry.

In this paper we elucidate, theoretically and experimentally, molecular motifs which permit Long-Lived Polarization Protected by Symmetry (LOLIPOPS). The basic assembly principle starts from a pair of chemically equivalent nuclei supporting a long-lived singlet state and is completed by coupling to additional pairs of spins. LOLIPOPS can be created in various sizes; here we review four-spin systems, introduce a group theory analysis of six-spin systems, and explore eight-spin systems by simulation. The focus is on AA'XnX'n spin systems, where typically the A spins are (15)N or (13)C and X spins are protons. We describe the symmetry of the accessed states, we detail the pulse sequences used to access these states, we quantify the fraction of polarization that can be stored as LOLIPOPS, we elucidate how to access the protected states from A or from X polarization and we examine the behavior of these spin systems upon introduction of a small chemical shift difference.

Accessing long-lived disconnected spin-1/2 eigenstates through spins > 1/2.

Pairs of chemically equivalent (or nearly equivalent) spin-1/2 nuclei have been shown to create disconnected eigenstates that are very long-lived compared with the lifetime of pure magnetization (T1). Here the classes of molecules known to have accessible long-lived states are extended to include those with chemically equivalent spin-1/2 nuclei accessed by coupling to nuclei with spin > 1/2, in this case deuterium. At first, this appears surprising because the quadrupolar interactions present in nuclei with spin > 1/2 are known to cause fast relaxation. Yet it is shown that scalar couplings between deuterium and carbon can guide population into and out of long-lived states, i.e., those immune from the dominant relaxation mechanisms. This implies that it may be practical to consider compounds with (13)C pairs directly bound to deuterium (or even (14)N) as candidates for storage of polarization. In addition, experiments show that simple deuteration of molecules with (13)C pairs at their natural abundance is sufficient for successful lifetime measurements.

Measuring long-lived 13C2 state lifetimes at natural abundance.

Long-lived disconnected eigenstates (for example, the singlet state in a system with two nearly equivalent carbons, or the singlet-singlet state in a system with two chemically equivalent carbons and two chemically equivalent hydrogens) hold the potential to drastically extend the lifetime of hyperpolarization in molecular tracers for in vivo magnetic resonance imaging (MRI). However, a first-principles calculation of the expected lifetime (and thus selection of potential imaging agents) is made very difficult because of the large variety of relevant intra- and intermolecular relaxation mechanisms. As a result, all previous measurements relied on costly and time consuming syntheses of (13)C labeled compounds. Here we show that it is possible to determine (13)C singlet state lifetimes by detecting the naturally abundant doubly-labeled species. This approach allows for rapid and low cost screening of potential molecular biomarkers bearing long-lived states.

Multicontrast nonlinear optical microscopy with a compact and rapid pulse shaper.

Homodyne detection can dramatically enhance measurement sensitivity for weak signals. In nonlinear optical microscopy it can make accessible a range of novel, intrinsic, contrast like nonlinear absorption and nonlinear phase contrast. Here a compact and rapid pulse shaper is developed, implemented, and demonstrated for homodyne detection in nonlinear microscopy with high-repetition rate mode-locked femtosecond lasers. With this method we generate two-photon absorption (TPA) and self-phase modulation images of gold nanostars in biological samples. Simultaneous imaging of two-photon luminescence and TPA also enables us to produce two-photon quantum yield images.

Accurately determining single molecule trajectories of molecular motion on surfaces.

This paper presents a method for simultaneously determining multiple trajectories of single molecules from sequential fluorescence images in the presence of photoblinking. The tracking algorithm is computationally nondemanding and does not assume a model for molecular motion, which allows one to determine correct trajectories even when a distribution of movement speeds is present. We applied the developed procedure to the important problem of monitoring surface motion of single molecules under ambient conditions. By limiting the laser exposure using sample scanning confocal microscopy, long-time trajectories have been extracted without the use of oxygen scavengers for single fluorescent molecules. Comparison of the experimental results to simulations showed that the smallest diffusion constants extracted from the trajectories are limited by detector shot noise giving error in locating the positions of the individual molecules. The simulations together with the single molecule trajectories and distributions of diffusion constants allowed us therefore to distinguish between mobile and immobile molecules. Because the analysis algorithm only requires a time series of images, the procedure presented here can be used in conjunction with various imaging methodologies to study a wide range of diffusion processes.

Micrometer-scale translation and monitoring of individual nanocars on glass.

Nanomachines designed to exhibit controlled mechanical motions on the molecular scale present new possibilities of building novel functional materials. Single molecule fluorescence imaging of dye-labeled nanocars on a glass surface at room temperature showed a coupled translational and rotational motion of these nanoscale machines with an activation energy of 42 +/- 5 kJ/mol. The 3 nm-long dye-labeled carborane-wheeled nanocars moved by as much as 2.5 mum with an average speed of 4.1 nm/s. Translation of the nanocars due a wheel-like rolling mechanism is proposed and this is consistent with the absence of movement for a three-wheeled nanocar analogue and the stationary behavior of unbound dye molecules. These findings are an important first step toward the rational design and ultimate control of surface-operational molecular machines.