3D Structure investigations in Pinna nobilis by...
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3D Structure investigations in Pinna nobilis by tomographic x-ray propagation imaging H. Metzger 1 , C. Olendrowitz 2 , R. Wilke 2 , M. Bartels 2 , T. Salditt 2 1 Max Planck Institute of Colloids and Interfaces, Depart. of Biomaterials, 14424 Potsdam 2 Inst. for X-ray Physics, Univ. G¨ ottingen, Friedrich-Hund-Platz 1, 37077 Gttingen, Germany Introduction: The aim of the proposed experiment was to analyze the complex microstructure of biogenic prismatic calcite crystals from the outer shell of a pinna nobilis mussel [1, 2, 3]. It is known that the strong interaction of organic macromolecules during the biogenic calcite growth results in improved mechanical properties of these crystals. In order to reveal the corresponding nanostruc- ture, previous high resolution x-ray diffraction on powdered specimens of calcite prisms from the Pinna nobilis revealed anisotropic lattice distortions, indicative of a complex mesostructure of in- organic crystalline domains within the organic network. Complementing these studies in Bragg diffraction, the present coherent imaging experiment was carried out to investigate this mesoscale structure, at a resolution high enough to characterize presumed nanosized building blocks compos- ing the mesocrystals, with respect to their size, shape and orientation. This information is needed to understand the role of the intra-crystalline organic network in the revealed microstructure. Sample are of prism shape with a diameter in the range of 20-50μm, and were studied by projection propagation imaging tomography. Phase contrast projection propagation imaging [4] and tomogra- phy are a full field imaging approach requiring scanning of only one (tomographic) axis, instead of scanning 3 degrees of freedom, as in 3D ptychographic coherent diffraction imaging (PCDI) [5]. crossed waveguide KB mirrors focal plane detector hologram pinna nobilis z1 z2 z y x A C D B E Figure 1: (A) Sketch of the in-line holographic tomography experiment. A monochromatic 13.8keV x-ray beam is focused by two KB-mirrors onto the system of two crossed planar waveguides (WG), serving as a virtual quasi-point source. The Pinna nobilis tip (sample Pinna300) is placed on a tomographic stage at a distance z 1 = 12mm behind the WG exit. (B) Typical image of the in-line optical microscope used for tomographic alignment and positioning of the sample. The area pixel detector (Maxipix) is placed at a distance z 2 + z 1 =5.29m away from the sample to record the magnified in-line hologram (Fresnel diffraction pattern). (C) Artifacts from the distorted wavefront of the KB remain after devision by the empty beam for illuminations by the direct KB beam (Sample PinnaNat), illustrating the need for an additional coherence filter (waveguide). (D) Projection image (hologram) after devision by the empty waveguide (WG) beam, recorded in 120 sec (scale bar 10 μm). The fringes at the facets of the biomineral indicate the high coherence of the setup. Phase contrast clearly dominates the contrast formation. (E) The volume texture of the WG- tomogram reconstruction with standard filtered backprojection algorithms was rendered using the AVIZO Fire software package. Images were recorded for 120 seconds in steps of 3 degrees spaced equiangular (no missing wedge). Using the Maxipix detector (pixelsize = 55μm) the geometric magnification (M = 418) results in an effective pixel size of 132nm. At the given resolution the proposed layered structure was not detectable.
Transcript of 3D Structure investigations in Pinna nobilis by...
3D Structure investigations in Pinna nobilis bytomographic x-ray propagation imaging
H. Metzger1, C. Olendrowitz2, R. Wilke2, M. Bartels2, T. Salditt2
1 Max Planck Institute of Colloids and Interfaces, Depart. of Biomaterials, 14424 Potsdam2 Inst. for X-ray Physics, Univ. Gottingen, Friedrich-Hund-Platz 1, 37077 Gttingen, Germany
Introduction: The aim of the proposed experiment was to analyze the complex microstructure ofbiogenic prismatic calcite crystals from the outer shell of a pinna nobilis mussel [1, 2, 3]. It is knownthat the strong interaction of organic macromolecules during the biogenic calcite growth results inimproved mechanical properties of these crystals. In order to reveal the corresponding nanostruc-ture, previous high resolution x-ray diffraction on powdered specimens of calcite prisms from thePinna nobilis revealed anisotropic lattice distortions, indicative of a complex mesostructure of in-organic crystalline domains within the organic network. Complementing these studies in Braggdiffraction, the present coherent imaging experiment was carried out to investigate this mesoscalestructure, at a resolution high enough to characterize presumed nanosized building blocks compos-ing the mesocrystals, with respect to their size, shape and orientation. This information is neededto understand the role of the intra-crystalline organic network in the revealed microstructure.Sample are of prism shape with a diameter in the range of 20-50µm, and were studied by projectionpropagation imaging tomography. Phase contrast projection propagation imaging [4] and tomogra-phy are a full field imaging approach requiring scanning of only one (tomographic) axis, instead ofscanning 3 degrees of freedom, as in 3D ptychographic coherent diffraction imaging (PCDI) [5].
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Figure 1: (A) Sketch of the in-line holographic tomography experiment. A monochromatic 13.8keV x-raybeam is focused by two KB-mirrors onto the system of two crossed planar waveguides (WG), serving asa virtual quasi-point source. The Pinna nobilis tip (sample Pinna300) is placed on a tomographic stageat a distance z1 = 12mm behind the WG exit. (B) Typical image of the in-line optical microscope usedfor tomographic alignment and positioning of the sample. The area pixel detector (Maxipix) is placed at adistance z2+z1 = 5.29m away from the sample to record the magnified in-line hologram (Fresnel diffractionpattern). (C) Artifacts from the distorted wavefront of the KB remain after devision by the empty beam forilluminations by the direct KB beam (Sample PinnaNat), illustrating the need for an additional coherencefilter (waveguide). (D) Projection image (hologram) after devision by the empty waveguide (WG) beam,recorded in 120 sec (scale bar 10 µm). The fringes at the facets of the biomineral indicate the high coherenceof the setup. Phase contrast clearly dominates the contrast formation. (E) The volume texture of the WG-tomogram reconstruction with standard filtered backprojection algorithms was rendered using the AVIZOFire software package. Images were recorded for 120 seconds in steps of 3 degrees spaced equiangular (nomissing wedge). Using the Maxipix detector (pixelsize = 55µm) the geometric magnification (M = 418)results in an effective pixel size of 132nm. At the given resolution the proposed layered structure was notdetectable.
Results: A projecton tilt series was recorded in a KB/waveguide optical setup, followed by directtomographic reconstruction of the raw data, after correction by the empty beam. The results show avery homogeneous density in the interior the prism, and hence give no indication for the presumedlayered structure. Two different samples of Pinna nobilis calcite crystals (prismatic layer of theshell) have been investigated, one native and one after annealing at 300 degree Celsius, denoted byPinnaNat and Pinna300 in the following. The working hypothesis was that annealing increases thecrystal size associated with a presumed platelet like structure. At first in line holograms have beenrecorded with the pure KB beam, since the high intensity on the order of 1011cps allows for veryfast tomographic data accumulation, with 10msec accumulation per frame (projection). These datahave been recorded with a high resolution detector system based on a 50µm thick LuAG : Ce scin-tillator screen imaged by an optical microscope (×4 objective) equipped with a CCD camera (PCO2000, 7.4µm pixel size), placed at z1 = 413.5mm. For a typical projection (sample PinnaNat),see Fig.1 (C). The problem with these data is that devision by empty KB beam is not sufficient toremove the artifacts from wavefront distortions of the KB beam. Suitable reconstruction algorithmsare currently under development, but not yet available.To provided a sufficiently clean wavefront the experimental strategy was adapted for further coher-ence filtering by x-ray waveguides. Now, the typical holograms are fully quantitative and almostfree of artifacts, see Fig.1(D) for a typical recording. 60 equally spaced angular projections (nomissing wedge) were recorded, each with 120s (net) counting time, in addition to empty beamrecordings. A typical projection (after devision by the empty beam) is shown in Fig.1. Aside fromthe strong facet (shape) signal, spots as well as corrugations are observed in the projection, whichcan be located by tomographic reconstruction. Before applying filtered back projection, it is desire-able to reconstruct the complex-valued exit wave and the corresponding transmission function ofthe object (phase and amplitude) from each holographic projection. This turned out to be a difficultissue, since the previously developed algorithms for phase contrast imaging in this setup [4] haveto be adapted for mixed phase and absorption contrast samples. A simple filtered backprojection(Ram-Lak Filter) of the raw data (after empty beam devision), however, gives a first impression ofthe 3D structure, and in particular of a internal architecture, Fig.1(E).
Conclusion: The tomographic reconstruction and rendering, shows that all visible corrugations arelocated solely on the surface of the calcite crystal. The existence of an internal structure composedof inorganic platelets and organic layers can not be proven, at least at the current resolution.
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