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Hermann Ehrlich,* Eike Brunner, Paul Simon, Vasily V. Bazhenov, Joseph P. Botting,
Kontantin R. Tabachnick, Armin Springer, Kurt Kummer, Denis V. Vyalikh,
Serguei L. Molodtsov, Denis Kurek, Martin Kammer, René Born, Alexander Kovalev,
Stanislav N. Gorb, Petros G. Koutsoukos, and Adam Summers
FULL PAPER
Calcite Reinforced Silica–Silica Joints in the Biocomposite
Skeleton of Deep-Sea Glass Sponges
palaeontologists, geologists, and biologists.
As the most basal metazoans, they are the
key to understanding the evolution of both
calcium- and silicon-based biomineralization. The manifestation of this mineralization, a skeleton of spicules embedded
in the body of the sponge, is typically a
complex arrangement of calcite or silica.
For example, the skeletal spicules of glass
sponges (Hexactinellida, Porifera) are valuable model systems for the investigation
of structure-function relationships in biomaterials, with the ultimate goal of identifying design strategies for new synthetic
materials.[1] Natural structural composites
often show a variety of desirable properties,
including transient properties in the presence of water.[2,3] The hexactinellid sponges
have silica skeletal structures that are both
flexible and tough,[1,4,5] because of their hierarchically layered
structure and the hydrated state of the silica.[6] Emblematic of
the complexity of sponge skeletons is Euplectella aspergillum,
in which the skeleton is an elaborate cylindrical latticelike
The hierarchically structured glass sponge Caulophacus species uses the
first known example of a silica and calcite biocomposite to join the spicules
of its skeleton together. In the stalk and body skeleton of this poorly known
deep-sea glass sponge siliceous spicules are modified by the addition of
conical calcite seeds, which then form the basis for further silica secretion to
form a spinose region. Spinose regions on adjacent spicules are then joined
by siliceous crosslinks, leading to unusually strong cross-spicule linkages. In
addition to the biomaterials implications it is now clear, from this first record
of a biomineral other than silica, that the hexactinellid sponges are capable
of synthesizing calcite, the ancestral skeletal material. We propose that, while
the low concentrations of calcium in deep sea waters drove the evolution of
silica skeletons, the brittleness of silica has led to retention of the more resilient calcite in very low concentrations at the skeletal joints.
1. Introduction
By virtue of their embedded, mineralized skeletons,
sponges are of great interest to materials scientists, chemists,
Dr. H. Ehrlich, Prof. Dr. E. Brunner, M. Kammer
Institute of Bioanalytical Chemistry
Dresden University of Technology
Bergstr. 66, D-01069 Dresden, Germany
E-mail: hermann.ehrlich@tu-dresden.de
Dr. P. Simon
Max Planck Institute of Chemical Physics of Solids
Noetnitzer Str. 40, D-01187 Dresden, Germany
V. V. Bazhenov
Institute of Chemistry and Applied Ecology
Far Eastern National University
Sukhanova 8, 690650 Vladivostok, Russia
Dr. J. Botting
Leeds Museum Discovery Centre
Carlisle Road,1, Leeds LS10 1LB, UK
Dr. K. Tabachnick
P.P. Shirshov Institute of Oceanology RAS
Nahimovski Prospect 36, 117312 Moscow, Russia
Dr. A. Springer, R. Born
MBZ and Institute of Materials Science
Dresden University of Technology
Budapester Str. 27, D-01069 Dresden, Germany
DOI: 10.1002/adfm.201100749
Adv. Funct. Mater. 2011, 21, 3473–3481
Dr. K. Kummer, Dr. D. V. Vyalikh
Institute of Solid State Physics
Dresden University of Technology
Zellescher Weg 16, D-01069, Dresden, Germany
Prof. Dr. S. L. Molodtsov
European XFEL GmbH
Notkestr. 85, D-22607 Hamburg, Germany
Dr. D. Kurek
Centre “Bioengineering” RAS, 7/1
Prospekt 60-ya Oktiabrya, 117312 Moscow, Russia
Dr. A. Kovalev, Prof. Dr. S. Gorb
Functional Morphology and Biomechanics
Zoological Institute
Christian-Albrecht-Universitaet zu Kiel
Am Botanischen Garten 9, D-24098 Kiel, Germany
Prof. P. Koutsoukos
Department of Chemical Engineering
University of Patras
1, Stadiou Str., GR-265 04 Patras, Greece
Prof. A. Summers
Friday Harbor Laboratories
University of Washington
620 University Road, Friday Harbor, WA. 98250, USA
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structure with at least six hierarchical levels of organization
spanning the length scale from nanometers to centimetres.[5]
The basic elements are laminated spicules that consist of a
proteinaceous axial filament surrounded by alternating concentric domains of consolidated silica nanoparticles and organic
interlayers.
Spicules assigned to hexactinellids are known from the earliest part of the animal fossil record (Ediacaran, 630 to 542 My).[7]
Their skeletons appear to have been optimized by natural selection to provide structural support, and as an inert scaffold for
various tissues encompassing a variety of functions. Organic
molecules including silicateins,[8] or more recently discovered
collagen[9] and chitin[10–12] are responsible for both the biosilicification and for the specific mechanical properties of their
skeletons. These molecules and their functions are currently
the subject of detailed discussions in modern chemical biology.
The skeletal structures of glass sponges are also remarkable
due to their size, durability, flexibility, and optical properties,[13]
however, numerous questions regarding the chemistry/biology
interface of these mostly unique constructions remain open.
Deep-ocean currents are generally perceived as sluggish,
but at intermediate depths (1000 to 5000 m) a is steady flow
is present, in which the current speeds up and slows down
as it encounters variations in terrain.[14] Sponges are sessile
organisms that filter great volumes of seawater to collect food
particles and resolved organic matter, and they rely partly on
ambient water currents; many are also raised above the sea floor
on spicular stalks in order to better exploit them. Photographs
of deep-sea hexactinellids, in natural conditions, demonstrate
remarkable flexibility and resilience of the stalks.[14] Because
the spicules are made from a brittle material this flexibility is
unexpected. The primary goal of the current study was to investigate the chemistry and the peculiarities of structural organization in the spicular stalk and body spicules of the hexactinellid
sponge Caulophacus species. (Figure 1).[15–17]
2. Results
2.1. Clublike Structures Within the Sponge’s Skeletal Framework
Caulophacus, including its subgenus C. (Caulophacus), are
mushroom-shaped sponges that are attached to hard substrates
by a long stalk. The atrial cavity is turned out, and represented
by the upper surface of the cap (Figure 1A,B). The tubular stalk
is primarily constructed from long diactine (two rayed) spicules
that are connected to their neighbors by numerous articulations
(Figure 1C–E) or by bridges (Figure 1F–G) through secondary
silica deposition. The stalk is fixed to the substratum by a basydictional plate, which contains mostly hexactines (six-rayed spicules) fused to each other at points of contact. The subgenus
is cosmopolitan, being distributed in all oceans and in some
seas with “normal” salinity at a depth of 130–6770 m. Currently
about 15 species are known.
Individual spicules were isolated from the Caulophacus sp.
stalk using pincers. We observed clublike constructions
(Figure 2A,B) at the ends of separated spicules using scanning
electron microscopy (SEM). These spinose, clublike structures
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were also observed in light microscopy images (Figure 2C).
To obtain more detailed information about localization of
these specific structures within the spicular articulations, we
employed gentle desilicification using a 2.5 M NaOH solution at
37 °C, as described previously.[9,10] After 14 days of immersion,
the clublike constructions could be observed as being located
within articulations using both light microscopy (Figure 2D)
and SEM (Figure 2E,F). Detailed analysis of the SEM images[18]
revealed that the spiny surfaces on these spicules could be
coupled with each other, preventing slippage against the corresponding silica-based articulation (Figure 3). This structure is
unique and evolved as a natural engineering solution to structural integrity problems in the construction of the hierarchical
skeletons of glass sponges.
Siliceous monaxon spicules with various spination patterns are well described in the sponge literature.[19] It has been
hypothesized that this phenomenon results from hypersilicification related to a high silica concentration in water.[20] However,
during alkali-based desilicification experiments we observed
that the spines of the clublike structures were highly resistant
to alkali treatment, in contrast to the surface silica layers, which
are deeply corroded under these conditions (Figure 3).
2.2. Identification of Calcite-Silica Composite
In order to proceed with more detailed studies, we mechanically
disrupted several spicules showing the clublike constructions, as
shown in Figure 4A. SEM images of these broken elements show
that the spines possess central structures, covered with a layer of
silica (Figure 4B). Elemental mapping (Figure 4C) and energy dispersive X-ray spectroscopy (EDX) (Figure 4D) unambiguously show
that calcium and not silicon is the main component of this central
structure (see the Supporting Information, Figure S1). Atomic
absorption spectroscopy[18] also confirmed the presence of calcium
in this spicular material, at an average concentration of 37.8 ±
9.0 ng mg−1 (mean ± standard deviation, s.d.), corresponding to
3.78 ± 0.90 mass percent of calcium in the whole spicule.
The chemical nature of the mineral phases within the
club-like structures was then investigated by means of
photoemission spectroscopy (PES) and X-ray absorption spectroscopy (XAS) (Figure 4E,F), performed on untreated spicules of Caulophacus sp. The appearance of Si 2p photoemission demonstrates the presence of silicon in all spicules, and
the exact position of the peak on the binding energy scale
grants an insight into its chemical bonding. Pure silicon is
observed at 100 eV bond energy, whereas SiO2 is found at
105 eV. SiOx compounds are observed between 100 and 105 eV
according to their stoichiometry (Figure 4E). The observed
bond energy of about 103 eV suggests that the silicon compound in the spicules has a silicium-to-oxygen ratio of 1:1,2.
Therefore we hypothesize that silicon oxide is present in
these skeletal structures in a bound form. This stoichiometry rules out foreign particles as a source of the silica signal.
Also, XAS spectra obtained for each of the sponge samples
(Figure 4F) show a strong signal at the Ca 2p absorption
edge. The fine structure of the near-edge X-ray absorption pattern closely resembles that of calcite. Transmission electron
microscopy (TEM) and electron diffraction analysis (EDA) of
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Figure 1. Complex organization of Caulophacus sp. spicular stalks. a) Caulophacus sp. on the rocky surface, 15 cm tall. b) The stalks of this sponge
possess a complex network of glassy spicules (c). d,e) Scanning electron microscopy (SEM) images show that spicules within stalks are connected
to each other with scaffoldlike articulations. f) SEM image that shows parallel spicules connected to each other by small crosslinks. g) Computer
reconstruction of these articulations.
the untreated clublike structures (Figure 5A,B) further confirm
these results. TEM diffraction analysis of the spicule shows
the presence of two phases, one amorphous (Figure 5C) and
one crystalline (Figure 5D). EDA for the spines shown as a
TEM image in Figure 5B correspond to the (0001) zone axis
of calcite with d spacings: 100 (1010) 4.32 Å; 010 (0110) 4.32
Å and 210 (2110) 2.49 Å. These data correlate well with those
reported previously for calcite identified in other invertebrates
using the electron diffraction technique.[21] The microstructure
of the central calcite crystals isolated by alkali treatment from
the clublike structures (Figure 5D) is also very similar to the
morphology of calcite crystal nuclei described previously[18,22]
(see Supporting Information, Figure S2). Further analysis
of the crystals (Figure 5E) isolated from clublike structures
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after alkali-based desilicification was achieved by infrared
(IR) (see the Supporting Information, Figure S3) and Raman
(Figure 5F) spectroscopy. The obtained FTIR spectra as well as
Raman spectra unambiguously confirm the calcite nature of
these crystals.
3. Discussion
Mechanical testing of the spicules suggests an important
strengthening role for the observed structures. Microindentation tests of the sponge architecture demonstrated only
slight plastic deformation (one micrometer or less), compared
with tens of micrometers of elastic deformation. Moreover,
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Figure 2. Clublike structures within the Caulophacus sp. skeleton. a,b) Isolated and broken stalk spicules, separated using pincers, showing clublike
structures. c) Clublike termination of a spicule observed using light microscopy. d) After immersion of the stalks in 2.5 M NaOH for two weeks, partial
dissolution of silica-based articulations revealed that club-like construction (arrows) are located within articulation joints. e,f) SEM images of the
partially dissolved articulations confirm the observations made using light microscopy.
buckling events were clearly seen on the indentation curves (see
the Supporting Information, Figure S4A). The sponge has rigid
outer layer (thickness 150 μm) where the spicules are interconnected more densely. This area has a reduced E-modulus
(REM) of 14.9 ± 4.5 MPa with the minimal measured value of
1.45 MPa. The bulk material of the sponge is softer and is relatively isotropic. The REM of the sponge in the axial direction
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was 614 ± 173 kPa, whereas in areas where single spicules protrude, it was just 3.82 ± 1.34 kPa (see the Supporting Information, Figure S4). The inner surface of the sponge has a reduced
number of cross-linking spicules and spicules with orientation
parallel to the surface, which explains the small REM values of
the innermost part of the sponge (REM 4.3 ± 0.5 kPa). However, after 100 μm of compression of the soft layer, the sponge
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Figure 3. Clublike spicules determining the structural integrity of Caulophacus sp. sponge stalks. a) SEM images show that clublike structures are
responsible for mechanical coupling between two thin spicules within one large spicule. b) Corresponding computer reconstruction of this coupling.
c,d) Alkali treatment led to dissolution of silica, allowing observation of the spines of clublike constructions using SEM, on the tenth day of immersion
in alkali. e,f) The spines seem to be resistant to alkali treatment in contrast to the weakly corroded silica-based material that covers the spicules and
forms the articulations.
material has REM of 618 ± 200 kPa (see the Supporting Information, Figure S4B), equaling that of the bulk sponge material.
The challenge now is to understand the mechanisms by
which this unique biocomposite is initially formed, at high pressure (420 ATM) and at low temperature (between 2 and 4 °C). In
most cases, calcification in sponges is confined to an extracellular space delineated by tightly adhering sclerocyte cells.[20] The
sclerocytes secrete a macromolecular matrix to the extracellular
space, which comprises a genetically programmed, threedimensional framework that guides spicule formation. It is difficult to differentiate between organically mediated and inorganic
precipitation, and a combination of different mechanisms in the
same species cannot be ruled out. A decisive factor for the control
of nucleation of calcium carbonate is the development of local
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supersaturation with respect to calcite or to any other calcium
carbonate polymorph. This in turn depends on the levels of calcium and carbonate ion activities. The latter depend strongly on
local pH as the dissolution of CO2 is favored at higher pH values.
Seawater may become supersaturated with respect to calcium
carbonate especially in spatially confined structures. The inner
space of the Caulophacus sp. spicule represents a microchannel
of approximately 1.0 μm in diameter flooded with ionic solution, while one extremity of the spicule is sealed by the silica. At
these conditions, contact with silicic surfaces is expected to favor
heterogeneous nucleation of calcite, as in this case lower supersaturation is needed in comparison with the value needed for
homogeneous. For example, calculation of the saturation of calcite at 2,000 meters, 5 °C and pH between 7.62 and 8.22 yielded
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Figure 4. Identification of mineral components within clublike constructions. a) The SEM image of a mechanically disrupted clublike spicule shows
that the spines each possess a nucleus that is covered by silica layer. b) Magnification of (a). Elemental mapping (c) and EDX analysis (d) show the
presence of calcium as the main component of this nucleus. e) Photoemission spectra of club-formed spicule showing the presence of two kinds of
silicon oxides. f) X-ray absorption spectra showing that calcium carbonate in the form of calcite is the second mineral component present within the
spicules.
values between 49 and 184, respectively.[23] Calcite is expected to
precipitate at a saturation exceeding 100, a value which corresponds for the ion activity of seawater to pH 8.00.
The observed morphology of this silica–calcite biocomposite
can be understood as the result of the crystallization of calcite crystals in the presence of polymeric silica.[24] Garcia-Ruiz
et al.[25] obtained numerous peculiar forms of alkaline-earth
metal carbonates in high-pH silica gel, which indicated that
silicate anions interact strongly with carbonates. Furthermore,
layers of silicates have been deposited on the surface of calcite,[26,27] a fact supporting crystallographic affinity for the two
inorganic solids. It has also been shown in vitro that a complex
crystalline architecture can be induced through the miniaturization of the growth subunits by covering the surface of carbonate crystals with silicate anions and by the self-organized
assembly of the miniaturized subunits.[28] The silica–calcite-
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based clublike structures made by Caulophacus sp. are the first
natural example of this principle, which confirms that biomineralization is a process in which organisms produce mineral
solutions for their own functional requirements,[29] starting on
nano- and microlevels of structural hierarchy.
Although poriferan skeletons may be composed of a large
variety of minerals, calcium carbonate and siliceous structures
very rarely co-occur in the same sponge. The only previously
known examples are in the form of solid calcareous skeletons
in addition to siliceous spicules, or as small calcareous spherules[30] or granules[31] in siliceous spiculate sponges. None of
these constitute biocomposites, and no sponge with calcareous
spicules has yet been found to secrete silica also.
It is less clear how the skeletons of early sponges were constructed, and this is of great significance in palaeontology and
early animal phylogeny. It has been argued that the different
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Figure 5. Calcite crystals within club-like structures. a) TEM image of the native Caulophacus sp. spicule with a fragment of club-like structure (arrow)
(B). Spicule chosen for selected area electron diffraction (SAED). c,d) TEM diffraction analysis of this spicule shows the presence of two phases:
amorphous with a diffuse amorphous halo at 2.05 Å (c) and crystalline (d). Electron diffraction pattern corresponding to the spines shown as a TEM
image in (b) corresponds to the calcite lattice with d spacings of 4.32 Å (101) and 2.49 Å (210). Crystals isolated after alkali-based desilicification[10] of
club-like structures are well visible using SEM (e). Their Raman spectra (f, above) are absolutely similar with those of calcite standard (f, below).
secretion mechanisms of spicules in Silicispongia (Demospongiae
and Hexactinellida) and Calcarea preclude homologous biomineralization between the sponge classes.[32] However, the heteractinid sponge Eiffelia globosa shows features characteristic of both
groups, and spicules have a bilayered structure suggestive of a
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silica–carbonate biocomposite.[33] The presence of a silica–calcite
composite in extant hexactinellids suggests that the mechanism of secretion is flexible in the precise structures formed,
and that extant and early sponges share a common biochemical
basis for secreting both materials. Although the structures of the
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skeletal elements themselves may have changed, as well as the
cytological architecture, the mineralogical secretion processes are
likely to be homologous. If similar structures to those in Caulophacus are confirmed in other taxa, the widespread presence of silica–calcite composites in the silicisponge lineage but not the calcareans, would tend to support an ancestral carbonate spicule mineralogy for Porifera. More data are needed from other sponge groups
before the full implications of such findings will be known.
4. Conclusions
Structural design of biological materials has evolved under
extreme evolutionary pressures to ensure the survival of a species, often in adverse environments.[34] We can now outline the
factors that are important for the highly deformable and resilient structure of the glass sponge stalk. Typically, slender rods
of small dimension are stiffer than expected.[17] Stress concentration near holes and notches is dissipated by materials with
microstructure such as the glass sponge stalk. In a foamlike
construction, a stiff and brittle material of single cells contributes to the volume, but not to the stiffness of the construction.
In foamlike sponge construction, the largest structural fibrelike elements appear to govern the fracture toughness and the
localization of microdamage. Twisting or bending of the fiberbased construction is an additional degree of freedom that dissipates stress. Additionally, clavate-shaped, spine-covered ends
of spicule, which are made of crystalline calcite, are responsible
for a mechanical interlocking of single spicule with each other.
A combination of two different materials at the level of single
spicule (see the Supporting Information, Figure S5), the interlocking system between spicules, and the fibrous composite
construction of the sponge stalk, make the entire construction
mechanically stable and simultaneously very flexible under
mechanically challenging environmental conditions. Intriguingly, the concept of turning weakness into strength based
on the university-diversity-principle (UDP),[34] in the case of
Caulophacus sp. sponge, can be discussed as very specific
because of the presence of two different inorganic materials.
In contrast to this siliceous sponge, diatoms, for example, can
transform a brittle biosilica into a ductile, strong, and tough
material, solely through alterations of its structural arrangement
at the nanoscale.[35,36] Thus, mechanical response of diatoms
cell walls can be greatly altered by simple alteration of their
structural geometry without the need to introduce new constituents.[35] Clearly, the future development of the comparative
computational models[37] of glass sponges and diatoms, as two
different biosilica-based and nanostructurally organized hierarchical systems, represents a scientific task of great interest.
The silica–calcite composite structures described here represent an additional example of multiphase biomineralizationphenomenon recently discussed by us.[38] Up to know, is is not
known why some organisms utilize, for example, silica rather
than calcium carbonate as a structural material. However, in
the case of extreme deep-sea environmental conditions, Mother
Nature decides to use both mineral phases within one very
complex designed skeletal network, which is definitively essential for biological survival of the fittest organism.
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5. Experimental Section
Sponge Samples: The investigated Caulophacus sp. specimens were
captured during the 22nd cruise of the R.V. “Academic Mstislav Keldysh”
in the N-E Pacific, off Bering Island, stn. 2316, 55 o 36,08-35’N 167 o 23,0424,46’ E, 4200-4294 m. Stalks of Caulophacus sp. were treated according
to the following procedure. Sponge material was stored for several
days in fresh sea-water. The sponge was dried afterwards for 4 days at
45 °C. Finally the sponge skeletal material was cleaned in 10% H2O2
and dried again at 45 °C. Tissue-free dried sponge material was washed
three times in distilled water, cut into 3 cm long pieces and placed in a
solution containing purified Clostridium histolyticum collagenase (Sigma)
to digest any possible collagen contamination of exogenous nature. After
incubation for 24 h at 15 °C, the pieces of glass sponge skeleton were
again washed three times in distilled water, dried and placed in a 15 mL
vessel containing chitinase solution (as described by us previously)[10] to
digest any possible exogenous chitin contaminations. After incubation
for 48 h at 25 °C, fragments of skeleton were again washed, dried, and
placed in 10 mL plastic vessels for storage and analysis.
Chemical Etching of Glass Sponge Skeleton: 8 mL of 2.5 M NaOH
(Fluka) solution was added to the 10 mL plastic vessel, which contains
fragments of the Caulophacus sp. stalks, purified as described above.
The vessel was covered and placed under thermostatic conditions at
37 °C without shaking. The same procedure was carrying out using
0.01% solution of HF (puriss. p.a., Fluka). The effectiveness of the alkali
desilicification procedures was also monitored using optical microscopy
and SEM at different locations along the length of spicular material and
within the cross-sectional area.
Atom Absorption Spectroscopy (AAS): A Varian SpectrAA-10
spectrometer with Varian GTA-96 graphite furnace atomizer was used to
determine the concentration of calcium within the purified samples of
Caulophacus. sp. spicules. Three samples were weighed into Eppendorf
microvials, dissolved by adding 100 μL HF to each sample and
homogenised for 30 s by shaking. Each sample was diluted with 900 μL
purified water and again homogenised. A wavelength of 239.9 nm was
used to measure absorption by calcium.
Photoemission Spectroscopy (PES) and X-ray Absorption Spectroscopy
(XAS): Measurements were performed at MAX-lab (Lund University,
Sweden) using radiation from the beamline D1011 bending magnet
dipole beamline located at the MAX II storage ring. X-ray absorption
spectra in the vicinity of the Ca 2p absorption threshold were recorded
in total electron yield (TEY) mode with a MCP detector. Photoemission
of the Si 2p core-level was obtained at 200 eV photon energy using a
SCIENTA SES200 electron energy analyzer.
Supporting Information
Supporting Information is available from the Wiley Online Library or
from the author.
Acknowledgements
We thank Prof. H. Lichte for use of the facilities at the Special Electron
Microscopy Laboratory for high-resolution and holography at Triebenberg,
TU Dresden, Germany. The authors thank R. Lakes, G. Wörheide,
G. Bavestrello for helpful discussions. The authors are deeply grateful to
R. Schulze, H. Meissner, G. Richter, O. Trommer for excellent technical
assistance. This work was partially supported by the DFG (Grant Nos. EH
394/1-1; MO 1049/5-1, ME 1256/7-1 ME 1256/13-1, and WO 494/17-1)
and by the BMBF (Grant No. 03WKBH2G), by a joint program “Mikhail
Lomonosov - II” of DAAD (Ref-325; A/08/72558) and RMES (AVCP Grant
Nr. 8066), and by Erasmus Mundus Co-operation Window Programme
2009. Part of this study was supported, as part of the European Science
Foundation EUROCORES Programme FANAS, by the German Science
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Received: April 4, 2011
Published online: July 29, 2011
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