Quantitative X-ray phase-contrast microtomography from a compact laser-driven betatron source

X-ray phase-contrast imaging has recently led to a revolution in resolving power and tissue contrast in biomedical imaging, microscopy and materials science. The necessary high spatial coherence is currently provided by either large-scale synchrotron facilities with limited beamtime access or by microfocus X-ray tubes with rather limited flux. X-rays radiated by relativistic electrons driven by well-controlled high-power lasers offer a promising route to a proliferation of this powerful imaging technology. A laser-driven plasma wave accelerates and wiggles electrons, giving rise to a brilliant keV X-ray emission. This so-called betatron radiation is emitted in a collimated beam with excellent spatial coherence and remarkable spectral stability. Here we present a phase-contrast microtomogram of a biological sample using betatron X-rays. Comprehensive source characterization enables the reconstruction of absolute electron densities. Our results suggest that laser-based X-ray technology offers the potential for filling the large performance gap between synchrotron- and current X-ray tube-based sources

Numerical approaches to time evolution of complex quantum systems

We examine several numerical techniques for the calculation of the dynamics of quantum systems. In particular, we single out an iterative method which is based on expanding the time evolution operator into a finite series of Chebyshev polynomials. The Chebyshev approach benefits from two advantages over the standard time-integration Crank-Nicholson scheme: speedup and efficiency. Potential competitors are semiclassical methods such as the Wigner-Moyal or quantum tomographic approaches. We outline the basic concepts of these techniques and benchmark their performance against the Chebyshev approach by monitoring the time evolution of a Gaussian wave packet in restricted one-dimensional (1D) geometries. Thereby the focus is on tunnelling processes and the motion in anharmonic potentials. Finally we apply the prominent Chebyshev technique to two highly non-trivial problems of current interest: (i) the injection of a particle in a disordered 2D graphene nanoribbon and (ii) the spatiotemporal evolution of polaron states in finite quantum systems. Here, depending on the disorder/electron-phonon coupling strength and the device dimensions, we observe transmission or localisation of the matter wave.Comment: 8 pages, 3 figure

X-Ray Phase-Contrast Tomography of Renal Ischemia-Reperfusion Damage

Purpose: The aim of the study was to investigate microstructural changes occurring in unilateral renal ischemia-reperfusion injury in a murine animal model using synchrotron radiation. Material and Methods: The effects of renal ischemia-reperfusion were investigated in a murine animal model of unilateral ischemia. Kidney samples were harvested on day 18. Grating-Based Phase-Contrast Imaging (GB-PCI) of the paraffin-embedded kidney samples was performed at a Synchrotron Radiation Facility (beam energy of 19 keV). To obtain phase information, a two-grating Talbot interferometer was used applying the phase stepping technique. The imaging system provided an effective pixel size of 7.5 mu m. The resulting attenuation and differential phase projections were tomographically reconstructed using filtered back-projection. Semi-automated segmentation and volumetry and correlation to histopathology were performed. Results: GB-PCI provided good discrimination of the cortex, outer and inner medulla in non-ischemic control kidneys. Post-ischemic kidneys showed a reduced compartmental differentiation, particularly of the outer stripe of the outer medulla, which could not be differentiated from the inner stripe. Compared to the contralateral kidney, after ischemia a volume loss was detected, while the inner medulla mainly retained its volume (ratio 0.94). Post-ischemic kidneys exhibited severe tissue damage as evidenced by tubular atrophy and dilatation, moderate inflammatory infiltration, loss of brush borders and tubular protein cylinders. Conclusion: In conclusion GB-PCI with synchrotron radiation allows for non-destructive microstructural assessment of parenchymal kidney disease and vessel architecture. If translation to lab-based approaches generates sufficient density resolution, and with a time-optimized image analysis protocol, GB-PCI may ultimately serve as a non-invasive, non-enhanced alternative for imaging of pathological changes of the kidney

The Gyc76C Receptor Guanylyl Cyclase and the Foraging cGMP-Dependent Kinase Regulate Extracellular Matrix Organization and BMP Signaling in the Developing Wing of <i>Drosophila melanogaster</i>

<div><p>The developing crossveins of the wing of <i>Drosophila melanogaster</i> are specified by long-range BMP signaling and are especially sensitive to loss of extracellular modulators of BMP signaling such as the Chordin homolog Short gastrulation (Sog). However, the role of the extracellular matrix in BMP signaling and Sog activity in the crossveins has been poorly explored. Using a genetic mosaic screen for mutations that disrupt BMP signaling and posterior crossvein development, we identify Gyc76C, a member of the receptor guanylyl cyclase family that includes mammalian natriuretic peptide receptors. We show that Gyc76C and the soluble cGMP-dependent kinase Foraging, likely linked by cGMP, are necessary for normal refinement and maintenance of long-range BMP signaling in the posterior crossvein. This does not occur through cell-autonomous crosstalk between cGMP and BMP signal transduction, but likely through altered extracellular activity of Sog. We identify a novel pathway leading from Gyc76C to the organization of the wing extracellular matrix by matrix metalloproteinases, and show that both the extracellular matrix and BMP signaling effects are largely mediated by changes in the activity of matrix metalloproteinases. We discuss parallels and differences between this pathway and other examples of cGMP activity in both <i>Drosophila melanogaster</i> and mammalian cells and tissues.</p></div

Matrix metalloproteinase and ECM involvement in <i>hh-Gal4 UAS-gyc76C-RNAi</i> phenotypes.

<p>(A-D’) Increased posterior MMP2::GFP levels (anti-GFP staining) in <i>gyc76C</i> knockdown wings (A-B’), compared with wild type (C-D’), at 24–25 hours AP (A,A’,C,C’) and 27–28 hours AP (B,B’,D,D’). The posterior increase was most obvious in apical focal planes (apical) and in projections of cross-sections summed along the proximo-distal axis of the image (X project.). (E) Ratios of posterior to anterior Mmp2::GFP intensity in apical portions of epithelia, corresponding to boxes in (A), in 23–25 hour AP Mmp2::GFP (Control) and <i>hh-Gal4 UAS-gyc76C-RNAi</i> (RNAi) wings. Error bars show standard deviation. The ratio was significantly higher in RNAi wings at p<0.001, using single-tailed Student’s T and Mann-Whitney tests. (F-I’) Increased anti-MMP1 levels in posterior veins and interveins of <i>gyc76C</i> knockdown wings at 23–28 hours AP, even as Trol levels decrease in posterior intervein pockets at 27–28 hours AP (F-H’). At 32–24 hours AP, when Trol is completely lost from intervein pockets, so is Mmp1 (I,I’). See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005576#pgen.1005576.s009" target="_blank">S9S–S9X Fig</a> for lower magnification images and anti-Mmp1 in control wings. (J,K) Rescue of PCV in <i>gyc76C</i> knockdown wings by expression of <i>UAS-TIMP</i>, as detected with anti-pMad (H) or anti-Trol (I). Ectopic pMad in proximal wing correlates roughly with the proximal region where abnormal levels of Trol accumulate. (L-P’) Rescue of PCV and ECM abnormalities in <i>gyc76C</i> knockdown wings by expression of <i>UAS-Mmp2-RNAi</i>. (L-N) Nearly normal PCVs, detected using anti-pMad (L,M) and anti-DSRF (N) at 24, 26 and 28 hours AP, respectively. (O-P’) Nearly normal anti-Trol (O,O’) and anti-LanB2 (P) staining in posterior veins and intervein pockets at 30 hours AP, although posterior has abnormal aggregates of 6G7 anti-CgIV staining (P’). (Q-T’) Effects of overexpression of <i>UAS-trol</i> (EP insertion into <i>trol</i> locus). (Q) Broadening of adult PCV caused by <i>hh-Gal4- UAS-trol</i>. (R) Rescue of adult PCV in <i>gyc76C</i> knockdown wing by <i>UAS-trol</i>, despite increased wing blistering. (S) Rescue of PCV in 34 hour AP <i>gyc76C</i> knockdown wing by <i>UAS-trol</i>, as assayed by downregulation of DSRF. (T,T’) Abnormal accumulation of diffuse 6G7 anti-CgIV staining proximal to PCV between L4 and L5 (T), and failure to rescue posterior intervein pockets (2.4x magnification detail, T’) after <i>UAS-trol</i> expression in 34 hour AP <i>gyc76C</i> knockdown wing.</p

Mapping of <i>gyc76C</i>^<i>3L043</i> and characterization of <i>gyc76C</i> vein phenotypes.

<p>(A) <i>3L043</i> genomic region. <i>3L043</i> failed to complement the deficiencies <i>Df(3L)Exel9061</i> and <i>gyc76C</i>^{<i>KG03723ex33</i>}. A bar marks a genomic region duplicated in the subset of the <i>iso-1</i> strain used to generate the Berkeley <i>Drosophila</i> Genome Project (BDGP) genomic sequence, but unlikely to be duplicated in the mutagenized strain [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005576#pgen.1005576.ref057" target="_blank">57</a>] (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005576#pgen.1005576.s002" target="_blank">S2 Fig</a>). (B) Domain structure of the Gyc76C protein, with an N-terminal, extracellular ANF receptor domain, a transmembrane (TM) domain, a putative phosphorylation site (phos), a kinase “dead” PTKc domain, a putative dimerization domain (dimer), and a C-terminal CYC domain. The L⁶³⁵H mutation of <i>gyc76C</i>^<i>3L043</i> is in the N-terminal region of the PTKc domain. (C) Conservation of Gyc76C’s L⁶³⁵ (red) in vertebrate NPR1s. (D) Wild type adult wing. (E) Crossveinless phenotype resulting from large posterior homozygous <i>gyc76C</i>^<i>3L043</i> mutant clones in <i>en-Gal4/UAS-FLP; gyc76C</i>^<i>3L043</i><i>FRT</i>^<i>2A</i>/<i>hs-GFP RpS17</i>^<i>4</i><i>FRT</i>^<i>2A</i> fly. (F) Crossvein and L5 disruption resulting from smaller <i>gyc76C</i>^{<i>KG0372ex33</i>} clones in <i>hs-FLP; gyc76C</i>^{<i>KG0372ex33</i>}<i>FRT</i>^<i>2A</i><i>/hs-GFP RpS17</i>^<i>4</i><i>FRT</i>^<i>2A</i> fly. (G,H) PCV disruption caused by <i>A9-Gal4</i>-driven (G) or <i>hh-Gal4</i>-driven (H) expression of <i>UAS-gyc76C-RNAi</i> (VDRC 6552). Detail in H shows wing with blistering phenotype typical of stronger Gal4 expression at 28°C. (I,J) Ectopic venation caused by <i>A9-Gal4</i>-driven (I) or <i>hh-Gal4</i>-driven (J) expression of <i>UAS-gyc76C</i>. Detail in (J) shows that the ectopic venation induced by <i>hh-Gal4</i> extends anterior to the A/P compartment boundary. (K) Pupal <i>hh-Gal4 UAS-GFP</i> wing at 24 hours AP showing GFP expression (green) posterior to the A/P compartment boundary and its position relative to the veins, marked with anti-pMad (red). (L) PCV disruption and lack of ectopic venation caused by <i>A9-Gal4</i>-driven expression of the cyclase dead <i>UAS-myc-gyc76C</i>^<i>D945A</i>.</p

For and Gyc76C are required for the refinement and maintenance of BMP signaling.

<p>(A,B) PCV regions of 24 hour AP wild type (wt) wings showing anti-pMad staining (A) and suppression of anti-DSRF expression (B) in veins. (C,C’) 24 hour AP <i>for</i>^<i>02</i> homozygote wing showing loss of loss of pMad (C) and failure to suppress DSRF (C’) in PCV. (D,D) 23 hour AP <i>hh-Gal4 UAS-gyc76C-RNAi</i> wings showing loss of pMad (D) but still slight suppression of DSRF (E) in PCV. (F) 28 hour AP <i>hh-Gal4 UAS-gyc76C-RNAi</i> wing showing failure to suppress DSRF in PCV. (G) Anti-pMad staining in 20 hour AP wild type wing. (H) 20 hour AP <i>en-Gal4 UAS-gyc76C-RNAi</i> wing showing abnormally broad anti-pMad in PCV and LVs. (I) 20 hour AP <i>for</i>^<i>02</i> homozygous wing showing pMad in PCV. (J-K’) Anti-pMad staining (red, white) in homozygous <i>gyc76C</i>^<i>3L043</i> (J,J’) or <i>gyc76C</i>^{<i>KG0372ex33</i>} (K,K’) clones (identified by absence of green GFP) in 28 hour AP <i>hs-FLP/+; gyc76C FRT</i>^<i>2A</i><i>/hs-GFP RpS17</i>^<i>4</i><i>FRT</i>^<i>2A</i> wings. Individual cells in PCV retain high levels of pMad (arrows), similar to levels in neighboring wild type PCV or LV cells. (L,L’) Normal anti-pMad staining (red, white) in PCV cells of homozygous <i>for</i>^<i>02</i> clone (identified by absence of green RFP) in <i>hsFlp</i>; <i>for</i>^<i>02</i><i>FRT</i>^40A/<i>ubi-RFP FRT</i>^<i>40A</i> 26 hour AP wing. (M) Anti-pMad (red) and anti-Myc (green) staining in a 24 hour AP <i>UAS-myc-gyc76C</i>/<i>+</i>; <i>hh-Gal4</i>/<i>+</i> wing. Arrow indicates ectopic pMad between L3 and L4 anterior to the PCV, outside the region of <i>hh-Gal4</i>-driven expression of Myc-Gyc76C. (N,O) Comparison of anti-pMad (red) staining in 25 hour +/<i>for</i>^<i>02</i> and <i>for</i>^<i>02</i>/<i>for</i>^<i>02</i> wings with <i>hh-Gal4 UAS-gyc76C-myc</i>/+ (anti-Myc, green). Ectopic pMad observed in <i>for</i> heterozygote (N) is lost in <i>for</i> homozygote (O).</p

Effects of <i>for</i> and <i>gyc76C</i> on wing ECM.

<p>Lines in (D-E”‘, L-L’) show approximate limits of posterior <i>hh-Gal4</i> expression. (A,A’) Anti-Trol (Perlecan) staining in control <i>for</i>^<i>02</i>/<i>CyO</i>, <i>Tb</i> wing at 28–30 hours AP. (B-C) Anti-LanB2 (B) and 6G7 anti-Collagen IV (CgIV) staining in control wild type wings at 28 hours AP (B) and 28–30 hours AP (C). Controls show normal ECM concentration in veins and intervein pockets in both anterior and posterior. (D-E”‘) Anti-Trol staining (D,D”), Trol-GFP (D’,D”‘), Anti-LanB2 (E,E”) and 6G7 anti-Collagen IV (CgIV) (E’,E”‘) staining in 32–34 hour AP <i>hh-Gal4 UAS-gyc76C-RNAi</i> wings. Posterior ECM is more diffuse in LVs and depleted from intervein pockets. In addition, Trol-GFP accumulates more strongly in L5 and PCV region, and is also found in vesicular puncta (arrows in D”‘) in interveins, and CgIV accumulated in abnormal aggregates in veins and interveins (E’,E”‘). (F-G’) 24 hours AP <i>trol-GFP; hh-Gal4 UAS-gyc76C-RNAi</i> wings. (F,F’) Posterior GFP puncta are visible in both basal and apical focal planes. (G,G’) High magnification images of wings stained with anti-Trol (red, white) and anti-FasIII (blue) to visualize cell outlines. GFP puncta are intracellular and do not stain with anti-Trol. (H,H’) Detail of posterior intervein region of <i>hh-Gal4 UAS-gyc76C-RNAi UAS-rab9</i>.<i>YFP</i> wing at 24 hours AP, showing abnormally large vesicles (H, DIC optics) that co-localize with Rab9.YPF fluorescence (H,H’). (I,I’) Anti-Trol staining in 28–30 hour AP <i>for</i>^<i>02</i> homozygote, aged and stained at same time as control in (A). Trol is broader and more diffuse in LVs, lost from PCV region, and depleted from intervein pockets. (J,K) Normal-appearing anti-Trol staining in <i>cv-c</i>^<i>1</i> (J) and <i>cv</i>^<i>43</i> (K) wings, except for loss of PCV. (L,L’) anti-Trol (L) or 6G7 anti-CgIV (L’) staining 28–30 hour AP <i>hh-Gal4 UAS-gyc76C-myc</i> wing. Intervein staining in the posterior is more diffuse and depleted from intervein pockets; this defect extends anterior to the region of <i>hh-Gal4</i> expression (lines) into intervein between L3 and L4 (arrow). Staining is normal anterior to L3.</p

PCV development and the genetic screen.

<p>(A) Model of signaling in the PCV. BMPs (Dpp and Gbb) secreted by adjacent LV cells bind to the Sog/Cv-Tsg2 complex, allowing movement into the PCV region. Cv-Tsg2 helps stimulate cleavage of Sog by the Tlr protease, freeing BMPs for signaling. Cv-2, largely bound to cells by glypicans, also binds BMPs, BMP receptors and Sog, locally transferring BMPs from Sog to the receptor complex. Cv-d also binds glypicans and BMPs and increases signaling by an unknown mechanism. (B) Diagram of vein and ECM development during the period of PCV formation in low magnification and high magnification cross-sections. As the dorsal and ventral epithelia reattach, the basal ECM of the early wing is remodeled, coming to lie in the vein channels and in scattered basolateral pockets between cells. Integrins and Dystroglycan concentrate at sites of basal-to-basal cell adhesion (both) and lining the veins (Dystroglycan). (C) Crossing scheme used to generate large homozygous mutant clones throughout the posterior compartment of the developing wing in heterozygous flies. <i>UAS-FLPase; FRT</i>^<i>2A</i> (3L) or <i>UAS-FLPase; FRT</i>^<i>82B</i> (3R) males were fed EMS and backcrossed to virgin females of the same genotype. <i>mut</i>* represents EMS-mutagenized chromosome. Virgin female F1 progeny were then crossed to <i>en-Gal4; FRT</i>^<i>82B</i>, <i>RpS3</i>^{<i>Plac92</i>}<i>Ubi-GFP / TM6</i> or <i>en-Gal4</i>; <i>hs-GFP RpS17</i>^<i>4</i><i>FRT2A /TM6</i> males. Approximately 50 females were used for each of the first two crosses. (D) <i>en</i> expression in posterior of late third wing discs, shown using the <i>en-lacZ</i> enhancer trap. (E) Large posterior homozygous clones, marked by the absence of GFP (white), induced in late third instar wing disc using <i>en-Gal4</i>/<i>UAS-FLPase</i>; <i>FRT</i>^<i>82B</i><i>/FRT</i>^<i>82B</i><i>RpS3</i>^{<i>Plac92</i>}<i>ubi-GFP</i>. (F) Test of the mosaic method using <i>en-Gal4</i>/<i>UAS-FLPase; FRT</i>^<i>82B</i><i>RpS3</i>^{<i>Plac92</i>}<i>ubi-GFP</i>/<i>FRT</i>^<i>82B</i><i>dystrophin</i>^{<i>EP3397</i>}; the adult wing shows the “detached” crossvein phenotype typical of <i>dystrophi</i>n (<i>dys</i>) loss.</p