Since its invention in 1930, Zernike phase contrast has been a pillar in optical microscopy and more recently in x-ray microscopy, in particular for low-absorption-contrast biological specimens. We experimentally demonstrate that hard-x-ray Zernike microscopy now reaches a lateral resolution below 30 nm while strongly enhancing the contrast, thus opening many new research opportunities in biomedicine and materials science.

We describe an approach for exploring microscopic properties of granular media that couples x-ray microtomography and distinct-element-method ͑DEM͒ simulations through image analysis. We illustrate it via the study of the intriguing phenomenon of instant arching in an hourglass ͑in our case a cylinder filled with a polydisperse mixture of glass beads that has a small circular shutter in the bottom͒. X-ray tomography provides three-dimensional snapshots of the microscopic conditions of the system both prior to opening the shutter, and thereafter, once jamming is completed. The process time in between is bridged using DEM simulation, which settles to positions in remarkably good agreement with the x-ray images. Specifically designed image analysis procedures accurately extract the geometrical information, i.e., the positions and sizes of the beads, from the raw x-ray tomographs, and compress the data representation from initially 5 gigabytes to a few tens of kilobytes per tomograph. The scope of the approach is explored through a sensitivity analysis to input data perturbations in both bead sizes and positions. We establish that accuracy of size-much more than positionestimates is critical, thus explaining the difficulty in considering a mixture of beads of different sizes. We further point to limits in the replication ability of granular flows away from equilibrium; i.e., the difficulty of numerically reproducing chaotic motion.

Glucose metabolism is difficult to image with cellular resolution in mammalian brain tissue, particularly with 18 fluorodeoxy-D-glucose (FDG) positron emission tomography (PET). To this end, we explored the potential of synchrotron-based low-energy X-ray fluorescence (LEXRF) to image the stable isotope of fluorine (F) in phosphorylated FDG (DG-6P) at 1 lm 2 spatial resolution in 3-lm-thick brain slices. The excitation-dependent fluorescence F signal at 676 eV varied linearly with FDG concentration between 0.5 and 10 mM, whereas the endogenous background F signal was undetectable in brain. To validate LEXRF mapping of fluorine, FDG was administered in vitro and in vivo, and the fluorine LEXRF signal from intracellular trapped FDG-6P over selected brain areas rich in radial glia was spectrally quantitated at 1 lm 2 resolution. The subsequent generation of spatial LEXRF maps of F reproduced the expected localization and gradients of glucose metabolism in retinal Mü ller glia. In addition, FDG uptake was localized to periventricular hypothalamic tanycytes, whose morphological features were imaged simultaneously by X-ray absorption. We conclude that the high specificity of photon emission from F and its spatial mapping at 1 lm resolution demonstrates the ability to identify glucose uptake at subcellular resolution and holds remarkable potential for imaging glucose metabolism in biological tissue. V V C 2012 Wiley Periodicals, Inc.

Serpins, a superfamily of protease inhibitors, control proteolytic cascades in many physiological processes. Genomic studies have revealed the presence of a high number of serpinencoding genes in Drosophila melanogaster, but their functions remain largely unknown. In a biochemical screen designed to detect protease inhibitors that may be implicated in early Drosophila development, we identified in embryos a ligand that forms a 67 kDa SDS-stable complex with the broad spectrum protease trypsin. Characterization of this ligand revealed it to be the recently described serpin, Spn5. Expression analysis by in situ and Northern blot hybridization indicated maternal transmission of the transcript as well as zygotic expression in many larval, pupal and adult tissues. Targeted repression by RNA interference did not alter early embryogenesis but resulted in a complete defect in the unfolding and expansion of the wings of freshly eclosed mutant flies, without other detectable effects on development.

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