Zinc and calcium are ubiquitous intracellular metals, and while a variety of quantitative probes have been developed for measuring intracellular changes in calcium concentration, the same is not true of zinc. We describe here the design, synthesis, and properties of the benzoxazole-based, ratiometric zinc probe, Zinbo-5. This bright fluorescent reporter has a quantum yield of 0.1 in the zinc-form, exhibits a Kd for Zn2+ in the nanomolar range, and shows significant changes in both excitation and emission maxima upon zinc binding. The utility of this cell permeable probe is demonstrated in fluorescence microscopy emission ratio imaging experiments on mammalian cells. We further show that Zinbo-5 is well suited for two-photon excitation microscopy ratio imaging and can readily reveal changes in intracellular zinc concentration within optical planes of single cells. To the best of our knowledge, this is the first example of two-photon excitation microscopy applied to ratio imaging of zinc. These methods can be applied to real-time emission or excitation ratio imaging studies of zinc physiology in living cells.

Summary Dynamic fluxes in the concentration of ions and small molecules are fundamental features of cell signaling, differentiation and development. Similar roles for fluxes in transition metal concentrations are less well established. Here we show that massive zinc fluxes are essential in the infection cycle of an intracellular eukaryotic parasite. Using single cell quantitative imaging we show that growth of the blood-stage Plasmodium falciparum parasite requires acquisition of thirty million zinc atoms per erythrocyte before host cell rupture, corresponding to a 400% increase in total zinc concentration. Zinc accumulates in a freely available form in parasitophorous compartments outside the food vacuole, including mitochondria. Restriction of zinc availability via small molecule treatment causes a drop in mitochondrial membrane potential and severely inhibits parasite growth. Thus extraordinary zinc acquisition and trafficking are essential for parasite development.

Analysis of monosaccharides is typically performed using analytical systems that involve a separation step followed by a detection step. The separation step is usually necessary because of the high degree of structural similarity between different monosaccharides. A novel sensing system for monosaccharides is described here in which living bacteria were designed to detect a model monosaccharide, L-arabinose, without the need for a separation step. In such sensing systems, analytes are detected by employing the selective recognition properties found in certain bacterial proteins. These systems are designed so that a reporter protein is expressed by the bacteria in response to the analyte. The concentration of the analyte can be related to the signal generated by the reporter protein. In the sensing system described here, the green fluorescent protein (GFP) was used as the reporter protein. L-Arabinose concentrations can be determined by monitoring the fluorescence emitted by the bacteria at 509 nm after excitation of GFP at 395 nm. The system can detect L-arabinose at concentrations as low as 5 x 10(-7) M and is selective over D-arabinose, the stereoisomer of the analyte, as well as over a variety of pentose and hexose sugars.

The pluripotency of human embryonic stem cells (hESCs) is important to investigations of early development and to cell replacement therapy, but the mechanism behind pluripotency is incompletely understood. Zinc has been shown to play a key role in differentiation of non-pluripotent cell types, but here its role in hESCs is directly examined. By mapping the distribution of metals in hESCs at high resolution by x-ray fluorescence microprobe (XFM) and by analyzing subcellular metal content, we have found evidence that loss of pluripotency is directly correlated with an increase in nuclear zinc. Zinc elevation not only redefines our understanding of the mechanisms that support pluripotency, but also may act as a biomarker and an intervention point for stem cell differentiation.

Single crystals of strontium barium niobate (Sr0.61Ba0.39Nb2O6), normally referred to as SBN:61, have been investigated using optical absorption, micro-Raman, photoluminescence, and electron paramagnetic resonance (EPR) techniques. Both undoped and cerium-doped (nominally 0.01 to 0.10% Ce) samples were included in the study. A broad optical absorption band centered near 490 nm and extending to the band edge near 380 nm is observed at room temperature in the Ce-doped crystals. Additional absorption, which we assign to iron, occurs just below the band edge in some of the samples. Photoluminescence data were taken at room temperature. Sharp emission bands at 545 and 650 nm are assigned to Er3+, an emission band at 755 nm is assigned to Ho3+, and a broader emission band extending from 700 to 850 nm is assigned to Cr3+. The SBN:61 crystals strongly absorb 9.4 GHz microwaves. To minimize the effect of these microwave losses, the EPR data were taken near 7 K. A broad isotropic EPR line due to Ce3+ ions is observed at g=0.9 in the Ce-doped samples. Additional isotropic EPR lines due to Fe3+ ions are observed at g=9.35 and g=4.10.