Nonlinear optical nanocrystals have been recently introduced as a promising alternative to fluorescent probes for multiphoton microscopy. We present for the first time a complete survey of the properties of five nanomaterials (KNbO(3), LiNbO(3), BaTiO(3), KTP, and ZnO), describing their preparation and stabilization and providing quantitative estimations of their nonlinear optical response. In the light of their prospective use as biological and clinical markers, we assess their biocompatibility on human healthy and cancerous cell lines. Finally, we demonstrate the great potential for cell imaging of these inherently nonlinear probes in terms of optical contrast, wavelength flexibility, and signal photostability.

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Transverse and longitudinal relaxation times (T1ρ and T1) have been widely exploited in NMR to probe the binding of ligands and putative drugs to target proteins. We have shown recently that long-lived states (LLS) can be more sensitive to ligand binding. LLS can be excited if the ligand comprises at least two coupled spins. Herein we broaden the scope of ligand screening by LLS to arbitrary ligands by covalent attachment of a functional group, which comprises a pair of coupled protons that are isolated from neighboring magnetic nuclei. The resulting functionalized ligands have longitudinal relaxation times T1(1H) that are sufficiently long to allow the powerful combination of LLS with dissolution dynamic nuclear polarization (D-DNP). Hyperpolarized weak “spy ligands” can be displaced by high-affinity competitors. Hyperpolarized LLS allow one to decrease both protein and ligand concentrations to micromolar levels and to significantly increase sample throughput.

Dynamic nuclear polarization (DNP) [1] combined with magic angle spinning (MAS) [2] can-under favorable conditions-enhance the nuclear spin polarization, that is, the difference between the populations of the Zeeman levels j ai and j bi of a spin I = 1 = 2 by up to two orders of magnitude (e DNP 10 2 ) with respect to the Boltzmann distribution at thermal equilibrium at ca. 100 K, while accelerating relaxation and hence reducing recovery delays by more than an order of magnitude (k = R DNP / R 1 = T 1 /t DNP > 10), thus providing a means of shortening measurement times by up to five orders of magnitude. Where suitable solvents can be found, typical enhancements for 13

Dynamic nuclear polarization (DNP) can enhance the nuclear polarization, that is the difference between the populations of the Zeeman levels j ai and j bi of spin I = 1/2, by up to four orders of magnitude with respect to their Boltzmann distribution at room temperature.[1] This enhancement arises from thermal mixing, which is brought about by microwave saturation of the EPR transitions of stable radicals that are mixed with the sample under investigation before freezing. In dissolution DNP, the sample is usually polarized at low temperatures and moderate magnetic fields (T = 1.2 K and B 0 = 3.35 or 5 T in our laboratory), [2] rapidly dissolved, [3] and heated to ambient temperature by a burst of water vapor.To minimize losses of nuclear spin polarization, the transfer from the polarizer to the NMR spectrometer or MRI magnet, including the settling of mechanical vibrations and convection currents, and, if required, the infusion into living organisms, must be completed within an interval T < T 1 . In our laboratory, the interval T has recently been lowered to 4.5 s. The radicals in the hyperpolarized solution lead to an increase of the longitudinal relaxation rate R 1 = 1/T 1 of the solute, thus limiting the timescales of the dynamic processes that can be monitored with hyperpolarized nuclei. A concomitant enhancement of the transverse relaxation rates R 2 = 1/T 2 leads to undesirable line-broadening. The relaxation rates R LLS = 1/T LLS of the populations of long-lived states (LLS) [4] and the decay rates R LLC = 1/T LLC of long-lived coherences (LLC) [5] are even more sensitive to the presence of free radicals than populations of eigenstates and single-quantum coherences. Free radicals can be toxic, and hyperpolarized solutions should not be infused into living organisms unless the radicals are removed.Herein, we demonstrate how N-oxide radicals that are widely used for DNP, such as 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), can be reduced by scavengers like sodium ascorbate (vitamin C) during the dissolution process into 2,2,6,6-tetramethylpiperidine-1,4-diol (TEMPOL-H; Scheme 1), thus extending transverse and longitudinal relaxation times of solutes and slowing down the decay of their polarization during and after transfer. Scavenging free radicals with ascorbate merely leaves ascorbyl radicals, which rapidly disproportionate, [6] in contrast to scavenging with thiol-based (DTT) or phenolic (Vitamin E) antioxidants, so that no paramagnetic species are present in the sample after dissolution and reduction.Scheme 1. The reduction of TEMPOL by sodium ascorbate, leading to the formation of a delocalized sodium ascorbyl radical and diamagnetic TEMPOL-H.