Naturally engineered designs exposing exceptional microstructural details and performance

article
Vesna Srot1, Birgit Bussmann1, Miloš Vittori2, Boštjan Pokorny3,4 and Peter A. van Aken1
1 Stuttgart Center for Electron Microscopy, Max Planck Institute for Solid State Research, Stuttgart, Germany
2 Department of Biology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia
3 Environmental Protection College + Eurofins ERICo, Velenje, Slovenia
4 Slovenian Forestry Institute, Ljubljana, Slovenia
 
Biography
Vesna Srot obtained her PhD in Geology from the University of Ljubljana, Slovenia. She continued with postdoctoral study at Max Planck Institute in Stuttgart, Germany. She is employed as a research scientist at Stuttgart Center for Electron Microscopy (StEM), Max Planck Institute for Solid State Research in Stuttgart, Germany. Her current research includes nanoscale analytical characterization of natural composite biomaterials and man-made bioinspired materials with emphasis on interfaces.
 
Abstract
A wide range of organisms produce materials which integrate organic matrix and crystalline or amorphous minerals in unique and complex constructions. Such natural composites show enhanced mechanical properties compared to their inorganic counterparts. Performance of functional materials is very often regulated at the nanoscale level. Advanced analytical and imaging transmission electron microscopy (TEM) techniques were employed to recognize innovative structural and compositional adaptations in abalone shell, claws of terrestrial crustaceans, human teeth and incisors of coypu. Ca-based phosphates and carbonates appear to be very common inorganic constituents of composites formed by living organisms. Use of analytical TEM methods (energy-dispersive X-ray spectroscopy: EDX; electron energy-loss spectroscopy: EELS) turned out to be crucial for investigations of such materials, where high resolution EELS demonstrated to be very useful tool for determination of CaCO3 polymorphs at high spatial resolution.
 
Acknowledgements
Thank you to: Ms. Ute Salzberger (MPI-FKF, Stuttgart, Germany) for tripod sample preparation, Dr. Julia Deuschle (MPI-FKF, Stuttgart, Germany) for nanoindentation measurements of coypu teeth, Ms. Felicitas Predel for SEM investigations and Mr. Bernhard Fenk for FIB sample preparation.
 
Corresponding author
Vesna Srot, Stuttgart Center for Electron Microscopy, Max Planck Institute for Solid State Research, Stuttgart, Germany
 
Introduction 
Living organisms have the exceptional ability to produce a variety of biominerals, consisting of inorganic and organic components, possessing diverse structure and composition. 
 
Fascinating and optimally assembled architectural designs reveal close linking of relatively simple crystalline or amorphous minerals and organic matrix that both together create natural functional composite materials. 
 
Although they are formed at ambient temperatures and atmospheric conditions, these materials show advanced characteristics[1,2], due to their construction solutions attained through evolution.
 
Their outstanding performance optimized for fulfilling different functions in animal bodies such as protection, motion, cutting and grinding, sensing, ion storage and many others, has been an inspiration for engineering materials with enhanced properties and smart design in the laboratory.   
   
Materials and Methods
 
Materials and TEM sample preparation
 
We studied unique microstructural and compositional adaptations in abalone shell[3], claws of terrestrial crustaceans[4], human teeth[5] and incisors of coypu[6]


As materials properties of functional composites are often controlled and regulated on the nanoscale, advanced analytical and imaging transmission electron microscopy (TEM) techniques were employed. TEM investigations of biological materials are critically fused with TEM sample preparation due to possible introduction of artefacts[7], as material is transformed from a living hydrated state to a dry state. Different TEM sample preparation techniques and their combinations were employed for our work: (i) mechanical thinning using a tripod polishing followed by subsequent ion-milling, (ii) ultramicrotomy and (iii) cutting a thin slab using a focused ion beam (FIB). 
(i) The combination of tripod polishing followed by Ar+ ion milling at lower voltages (500-1000 V) while cooling the sample with liquid nitrogen (L-N2) was successfully applied for preparing TEM lamellae of different natural composite materials where higher amounts of harder inorganic crystalline or amorphous components are combined with an organic matrix[3,5,6].
(ii) Ultramicrotomy delivered better results for samples prepared from bulk dentine in teeth where the density of dentinal tubules was higher. Similarly, we observed great benefits of such sample preparation when applied to other natural composite materials containing higher organic content[4-6]
(iii) Detailed microstructural observations have revealed smaller voids in crystalline components (aragonite platelets in abalone shell and hydroxyapatite crystals in teeth[3,5]) only for FIB prepared samples. Therefore it is plausible to believe that such microstructural features are artefacts produced by FIB preparation and thus such samples have not been used for our detailed investigations. 


Our findings emphasize the importance of TEM sample preparation and its potential impact on investigations of natural composites. Such materials are exceptionally demanding for both, TEM sample preparation and characterization, due to their highly complex microstructures and chemistries. 
 
Methods 
High-angle annular dark-field scanning TEM (HAADF-STEM) imaging combined with energy-dispersive X-ray spectroscopy (EDX) and electron energy-loss spectroscopy (EELS) measurements were carried out at 200, 80, 60 and 30 kV with an advanced analytical TEM/STEM (JEOL ARM200F, JEOL Co Ltd.) microscope, equipped with a cold field-emission gun and a DCOR probe Cs-corrector (CEOS Co Ltd.). EELS elemental maps (2D spectrum images) were obtained in STEM mode with a post-column energy filter with high-speed dual-EELS acquisition capability (Gatan GIF Quantum ERS, Gatan Inc. Pleasanton, USA). EDX spectra and elemental profiles were obtained by acquiring area and line scans using a 100 mm2 JEOL Centurio SDD-EDX detector and the Thermo Noran System 7 EDX system (Thermo Fisher Scientific Inc.).
 
High-resolution TEM (HRTEM) and electron diffraction experiments were performed at 200, 80 and 60 kV with an advanced TEM (JEOL ARM200F, JEOL Co Ltd.), equipped with a cold field-emission gun and a CETCOR image corrector (CEOS Co Ltd.).


Energy-loss near-edge structures (ELNES) and low-loss EELS measurements at high energy resolution were done with the Zeiss SESAM microscope (Zeiss, Oberkochen), a 200 kV FEG TEM/STEM microscope equipped with an electrostatic Ω-type monochromator and the MANDOLINE filter[8]. EEL spectra were obtained in image mode with a dispersion of 0.037 eV/channel. From each of the different areas 50 to 100 spectra were acquired and afterwards aligned and averaged using an in-house developed script for Digital Micrograph (Gatan, Inc.). 


Energy-filtered TEM (EFTEM) images were recorded at energy losses from 22 to 73 eV with an energy-loss increment of 3 eV and an energy-selecting slit of 3.41 eV by using in-house developed software (a script written for Digital Micrograph). 
 
Bright field (BF-) and annular dark field (ADF)-STEM imaging combined with EELS and EDX was carried out in a VG HB501UX microscope, which is a dedicated STEM instrument operated in ultra-high vacuum at an accelerating voltage of 100 kV, equipped with a cold field-emission gun (FEG) source and an EELS (Gatan UHV Enfina system) spectrometer. 
 
Background subtraction of the EEL spectra was performed by the power law method[9]. At every position, simultaneous thickness measurements were performed by acquiring low-loss spectra. The relative thicknesses (t/λ) were determined from the low-loss EEL spectra using the routine implemented in Digital Micrograph, where t is the absolute sample thickness and λ the inelastic mean free path.
 
Quantitative EDX analysis was done using experimentally determined k-factors measured from standard specimens under the same experimental conditions as for the measured natural samples.
 
Results / Discussion
 
Interfaces in natural composite multilayers in abalone shell
 
Mollusk shells appear in many diverse shapes and sizes, but surprisingly the prominent building material is for the most cases calcium carbonate (CaCO3), primarily in the form of the two polymorphs, aragonite and/or calcite. Interestingly, when both polymorphs are present in the same shell, they are always spatially separated in different sectors[10-11].
 
The shell acts as armour having a fundamental function to protect the soft body of the animal[12]. The structure of typical abalone shells consists of an outer prismatic calcite layer and an inner nacreous aragonite layer. Polygonal aragonite platelets separated by thin protein-polysaccharide organic matrix[10] are composing inorganic/organic composite material called nacre or the mother-of-pearl. Although the organic matrix represents only up to 5 wt% of nacre, it is of essential importance for the mechanical properties of this multilayered composite[10,13].        
 
The abalone shell (Haliotis) used in our study[3] is shown in Figure 1A. The shell was damaged during growth but afterwards self-repaired due to astonishing healing mechanisms. An interface between the outer prismatic calcite (CA) and inner nacreous aragonite (AR) shown in the BF-STEM image (Fig. 1B) appears to be abrupt. Based on our observations, various morphological appearances of different CaCO3 polymorphs in abalone shell make their visual distinction not possible. Therefore analytical EELS investigations at high energy and high spatial resolution are mandatory.
 
 

Figure 1 (A) Photograph of an abalone shell used in our study. (B) BF-STEM image from an interface region between nacreous aragonite (AR) and prismatic calcite (CA). A polarized light optical micrograph is shown in the inset. (C,D) Ca-L2,3 and O-K ELNES obtained from AR and CA regions. (E) Polarized optical micrograph of a self-healed area. Arrow indicates position of EFTEM map in (G). (F) The integral intensity of the Ca-M2,3 edges highlighted in spectra (grey shaded areas) from the low-loss energy region were used to form the EFTEM map shown in (G). (H) ADF-STEM image of nacre that is forming a multilayered structure consisting of alternating aragonite platelets and organic layers. (I) ADF-STEM image of magnified aragonite-organic interface in nacre with corresponding C-K and Ca-L2,3 ELNES (J) acquired from aragonite platelet (1), mineral bridge (2) and organic layer (3). 
 
The Ca-L2,3 and O-K ELNES collected from calcite and aragonite are displayed in Figures 1C and 1D, respectively. Very obvious differences between the Ca-L2,3 ELNES of aragonite and calcite appear due to the different coordination environment of calcium, where calcium atoms in calcite are six- and in aragonite nine-fold coordinated[14]. The two main spin-orbit split L3 and L2 peaks, also called white lines, are characteristic for the Ca-L2,3 ELNES of calcite and aragonite. The fundamental difference that can be used as a fingerprint to distinguish calcite and aragonite polymorphs is the intensity of the pre-peaks (marked by the arrows in Fig. 1C); they appear much weaker in aragonite compared to calcite and are shifted closer to the main L3 and L2 maxima. The observed intensity difference of the pre-peaks arises from the different coordination environment of the calcium atoms. Furthermore, also the O-K ELNES can be beneficially applied for polymorph determination, as it shows very distinct fine spectral features. The sharp feature at around 534 eV is followed by three clearly resolved peaks in calcite and two features in spectra from aragonite (Fig. 1D). 
 
Self-healing is a quite common phenomenon in nature, allowing organisms to self-repair after an injury[12]. A polarized light optical micrograph from an area across the healed part of abalone shell is displayed in Figure 1E. The intensity of the background-subtracted Ca-M2,3 edges (grey shaded area in Fig. 1F) in the energy range between 28.2-30.6 eV was used to form an EFTEM image (Fig. 1G). The calcite-aragonite interface appears to be relatively rough, with an organic layer extending even in calcite. Organisms very often form CaCO3 as calcite and/or aragonite polymorphs. Since this material is usually very beam sensitive, the Ca-M2,3 edges that have excitation energies in the low-loss EELS range where acquisition times are much reduced due to a high scattering cross section yielding a high EELS signal, are a very elegant alternative to distinguish those polymorphs at the nanoscale without introducing any artefacts due to electron beam damage. 
 
Nacre is a natural multi-layered composite material with mechanical properties, especially strength and toughness, which are superior to pure mineralogical counterpart due to particularly efficient arrangement[1,2].  The aragonite platelets forming the inorganic layers in nacre are vertically connected via mineral bridges through the organic layers, as reported in previous studies[15,16]
 
We have investigated the mineral–organic interfaces in nacre by means of imaging and analytical STEM. An ADF-STEM image of a nacreous multilayer with an alternating stacking of aragonite platelets separated by organic layers and an ADF-STEM image of the mineral bridge intersecting the organic layer and linking neighbouring aragonite platelets are displayed in Figures 1H and 1I. The C-K and Ca-L2,3 ELNES (Fig. 1J) acquired from the aragonite platelet and the mineral bridge show no considerable difference in their spectral fine structure, whereas the C-K ELNES from the organic layer is remarkably dissimilar. Based on these facts we can confirm the aragonite structure of mineral bridges in abalone shell.      
 
Structural anisotropy in crustacean claws
 
Claws of the final articles (dactyli) of walking legs in the terrestrial crustacean Porcellio scaber are thin skeletal structures supporting the whole body. Their design and mineral composition are functionally optimized, since they are predominantly subjected to heavy unidirectional loads, as the claws are loaded predominantly in the axial direction, and wear. The microscopic architecture and chemistry of P. scaber claws were analysed [4].
 
A scanning electron microscopy image (SEM) of the walking leg dactylus claw is presented in Figure 2A. The exoskeleton forming the claws is a fine-tuned composite incorporating chitin fibres and mineral particles. On a larger scale, it incorporates a mineralized inner layer (the endocuticle) and a non-mineralized outer layer (the exocuticle), which was demonstrated by EDX to be highly brominated, especially towards the tip (Fig. 2B). A diagram picturing the components of the dactylus is shown in Figure 2C.
 
Figure 2 (A) SEM image of the walking legs in Porcellio scaber. (B) EDX spectrum showing presence of Br measured in non-mineralized exocuticle. (C) Diagram of the dactylus claw. (D) HAADF-STEM image of endocuticle from dactylus base containing calcite particles with corresponding SAED pattern (inset). (E) HAADF-STEM image of the fibrous mineral particles in the claw endocuticle with corresponding SAED pattern (inset). (F) EDX, (G) Ca-L2,3 ELNES and (H) O-K ELNES showing that the chemical composition of endocuticle from dactylus base corresponds to calcite (CA) and from the claw endocuticle to amorphous calcium phosphate (ACP).
 
Visualization of the mineralized dactylus base (Fig. 2D) combined with selected area electron diffraction pattern (SAED) (Fig. 2D inset) and analytical STEM EDX (Fig. 2F) and EELS (Fig. 2G, 2H) confirms calcite as the predominant mineral in both endocuticle and exocuticle of the dactylus base. The Ca-L2,3 and O-K fine structural details recorded from the claw dactylus base show close resemblance to the spectra recorded from pure calcite[3], confirming the presence of the calcium carbonate polymorph calcite. In the mineralized endocuticle of the claw, the chitin-protein fibers are closely intertwined with mineral particles forming an anisotropic material providing maximum strength in the direction of loading (Fig. 2E).
 
Surprisingly, by combining our analytical STEM (Fig. 2F-H) and SAED (Fig. 2E inset) measurements we have proven that the mineral in the claw endocuticle is amorphous calcium phosphate and not calcite, which ordinarily dominates in crustacean exoskeletons. The incorporation of amorphous calcium phosphate and a non-mineralized bromine-enriched outer sheath are very likely features that increase the fracture resistance of the claws.   
 
Chemistry and microstructure controlled tissues in human teeth
 
The inorganic calcium phosphate component hydroxyapatite (HA), closely associated with the organic component, mainly collagen, forms composite mineralized tissues in human bodies. Biological hydroxyapatite is compositionally and structurally very sophisticated, and interestingly, it never appears as pure hydroxyapatite, but rather as chemically precisely adjusted Ca-deficient carbonate-containing apatite modified mainly with magnesium, sodium, potassium and zinc[10].
 
Teeth consist of three unique hard dental tissues (marked in Fig. 3A) – enamel (EN), dentine (D) and cementum (C), where each of them possess different physical properties and are together smartly combined into one functional apparatus. Enamel is a highly mineralized tissue, consisting of 96 wt% of inorganic material, displaying extremely long, close packed and highly oriented hydroxyapatite crystals (Fig. 3B). Dentine (Fig. 3C) forms the bulk of the teeth. The content of organic material in dentine is considerably higher (~ 20 wt%) compared to enamel[10] which reflects in the relatively softer and heterogeneous microstructure of dentine. Corresponding overview EEL spectra obtained from enamel and dentine areas containing P-L2,3, C-K, Ca-L2,3, N-K and O-K edges are shown in Figure 3D. Notably the intensive C-K edge in the spectrum from dentine stems from high concentration of organic material present in dentine. 
 
Figure 3 (A) Optical micrograph of a longitudinal cross-section of the human permanent maxillary premolar with marked positions of enamel (EN), dentine (D) and cementum (C). BF-STEM image of enamel (B) and dentine (C). (D) EEL spectrum acquired in the energy range of P-L2,3, C-K, Ca-L2,3, N-K and O-K edges from enamel (EN) and dentine (D). (E) BF-STEM image showing dentinal tubules (DT) separated by intertubular dentine (ID) and surrounded by peritubular dentine (PD). (F) In ADF-STEM image and enlarged area is presented. (G) Ca/P – Mg/P at% ratio determined from EDX spectra that were measured from EN, ID and PD.
 
Dentinal tubules (DT) are the most prominent structures that penetrate through dentine and are directly responsible for its permeability[17]
 
A low-magnification BF-STEM image obtained from an area with high density of DTs is shown in Figure 3E. An enlarged view of the rim of dentinal tubules clearly shows the wall of the mineralized peritubular dentine (PD) that is directly surrounding the dentinal tubules (Fig. 3F).  Peritubular dentine appears relatively dense compared to intertubular dentine (ID) that forms the bulk between the dentinal tubules; in addition the thin elongated needle shaped hydroxyapatite crystals occur smaller in peritubular dentine compared to the ones in intertubular dentine. 
 
Average Ca/P at% ratios determined by analytical EDX measurements of enamel and intertubular dentine are rather identical; however there is an obvious decrease in the Ca/P at% ratio combined with an increase in the amount of Mg in peritubular dentine (Fig. 3G). Unexpectedly, the Mg/P at% ratio determined from peritubular dentine is greatly higher than the value obtained from intertubular dentine and is closely associated with lower Ca/P at% ratio. From a crystal chemistry point of view our data imply incorporation of Mg into the hydroxyapatite lattice by occupying Ca positions. The presence of smaller hydroxyapatite crystals in peritubular dentine compared to intertubular dentine is presumably linked to a higher Mg concentration, which closely agrees with previously existing studies[18].   
 
Iron-enriched incisors  
 
Rodent´s incisors are a brilliant example of natural complex organic-inorganic composite material with smartly adjusted design and precisely tailored and controlled chemistry. Only the front part of long continuously growing incisors is covered by hard and resistant enamel, while softer dentine forms the bulk of the teeth and acts as a mechanical support for enamel. Due to heavy gnawing loads the bulk dentine is being gradually removed, and hence remodelling the incisor into a self-sharpening device (Fig. 4A). The surface of incisors of some rodent species shows characteristic orange-brown colour appearing due to the presence of iron[19].
 
Figure 4 (A) Bottom incisor of a coypu showing a sharp edge. (B) ADF-STEM image of the interface between pigmented enamel (Fe-EN) and Fe-rich surface layer (Fe-SL). (C) EDX concentration data for Fe, Ca and P measured from over 500 different positions and sorted by Fe concentration. (D) O-K ELNES recorded from different positions within the Fe-SL showing differences due to different Fe concentration. (E) EFTEM image of the interface between Fe-SL and Fe-EN showing overlapped Ca (blue) and Fe (red) chemical maps. (F) HAADF-STEM image of the hydroxyapatite crystals (HA) surrounded by ferrihydrite pockets (Fh) with corresponding Fe (red) and Ca (blue) EELS elemental maps (G). (H) O-K ELNES obtained from ferrihydrite pocket (red) and hydroxyapatite crystals (blue). HAADF-STEM images of pigmented (I,J) and non-pigmented enamel (K,L).
 
Building strategies and compositional adjustments in incisors of coypu (Myocastor coypus) were explored recently[6]. Nanoscale chemical analysis indicated the presence of iron (Fe) in the outer pigmented enamel (Fe-EN), while predominantly calcium (Ca) and phosphorous (P) as major elements of hydroxyapatite were detected in the inner enamel (EN).
 
A magnified STEM image of the upper incisor´s surface (Fig. 4B) exposed an additional thin layer covering pigmented enamel, in which we detected an unusually high and variable Fe concentration.
 
Further detailed analysis uncovered three loosely separated Fe-containing amorphous phases based on the different Fe content (Fig. 4C).
 
As the Fe-L2,3 ELNES can give insights into the local bonding, electronic structure and iron oxidation state, Fe-containing compounds were characterized through their fine structural details of the O-K and Fe-L2,3 ELNES.
 
The Fe-L2,3 ELNES (Fig. 4C inset) recorded in the Fe-rich surface layer (Fe-SL) shows a strong pre-peak on the low-energy side of the Fe-L3 line which is characteristic of trivalent iron, suggesting a predominantly 3+ oxidation state for Fe in the Fe-rich surface layer.
 
The O-K ELNES exhibits three prominant, to the Fe concentration linked spectral features (Fig. 4D). The O-K ELNES of various known Fe oxides, Fe hydroxides and Fe phosphate containing Fe in the 3+ valence state, Ca phosphates and amorphous Ca phosphate (ACP) were used as fingerprints and compared to the O-K ELNES obtained from the Fe-rich surface layer. The gradual decrease of Fe related with an increase in Ca and P concentrations is closely reflecting by the changes of the O-K ELNES.
 
We have demonstrated that three loosely separated phases, namely ferrihydrite, (Fe,Ca)-phosphate and (Ca,Fe)-phosphate and their intermixtures are constituents of the Fe-rich surface layer. The typical orange-brown colour of rodent´s incisors is therefore closely related to the presence of the Fe-rich surface layer.
 
An energy-filtered TEM (EFTEM) image (Fig. 4E) of the rough interface between Fe-rich surface layer and pigmented enamel was formed by overlapping the Ca (blue) and Fe (red) chemical maps obtained by using the background-corrected signal of the Ca-M2,3 and Fe-M2,3 edges. Interestingly, the base of the Fe-rich surface layer always starts with ferrihydrite (Fh) and either stays as ferrihydrite or alters into (Fe,Ca)- or (Ca,Fe)-phosphate. In the underlying outer, pigmented enamel, Fe-rich material was recognized in the spaces surrounding the needle-shaped hydroxyapatite crystals that are arranged with a long axis towards the tooth surface.  A view along the long axis of the hydroxyapatite crystals in the HAADF-STEM image (Fig. 4F) combined with the Fe and Ca maps obtained from EEL spectrum images (SI) (Fig. 4G) clearly reveals the presence of two separated phases. A chemically different Fe-rich phase containing Fe in the 3+ valence state, filling the pockets surrounding the hydroxyapatite crystals, was assigned through fine spectral features of the O-K edge as ferrihydrite (Fig. 4H). 
 
Rodents’ incisors are subjected to extremely harsh mechanical conditions. Our measurements of the mechanical properties[6] revealed an important protection function of the relatively soft Fe-rich surface layer that seems to soften the pressure applied to teeth during gnawing. Microstructural investigations of pigmented and non-pigmented enamel demonstrated a direct connection between the microstructure and material properties of incisors.
 
Close arrangement of hydroxyapatite crystals and ferrihydrite in pigmented enamel is creating dense and mechanically advanced material (Figs. 4I-J). On the other hand, in non-pigmented enamel spaces between hydroxyapatite crystals are not filled (Figs. 4K-L), resulting in lower material performance compared to pigmented enamel. As expected, dentine is (due to much higher organic content) relatively soft and can be removed at much higher rate than hard enamel during gnawing, creating a sharp cutting edge.
 
Our observations show a close link between the microstructure, chemistry and performance in rodent´s incisors. Their special functional design involves components possessing unique microstructure and chemistry, enable them to ensure structural stability on a long term basis.
 
Summary and conclusions
 
Natural composite materials combine ideal architectural design, finely tuned hierarchical microstructure, surprisingly simple compounds and high functionality.
 
Although microstructural and compositional adaptations of materials often appear relatively simple and nonessential, they have important influence on materials´ characteristics. 
 
The morphology, orientation and phase transformation of inorganic crystal phase are controlled and influenced through the synergy with the organic matrix.
 
In different skeletal elements, the organic matrix is very often closely linked with the Ca-based inorganic minerals, carbonates and phosphates, forming unconventional architectures starting at a level that need to be, due to their intertwined structure, investigated on the nanoscale at high spatial and high energy resolution in order to understand how they affect the mechanical properties.
 
In this paper, our investigations by using advanced imaging and analytical (S)TEM techniques for understanding such composite materials are presented for various functional naturally formed organic-inorganic compounds. 
 
Living organisms have the ability to combine and precisely harmonize organic and inorganic components with ordinary properties into building masterpiece composite materials with exceptional performance.
 
Although materials adaptations might appear as minor, they have enormous impact on materials functionality. Very often the research of such natural designs can serve to inspire man-made designs imitating advanced materials properties with applications in technology and medicine as well as in ordinary human life.
 
References
 
[1] U.G.K. Wegst and M.F. Ashby, The mechanical efficiency of natural materials, Phil. Mag. 84, pp. 2167-2181, 2004.
[2] A.P. Jackson and J.F.V.J. Vincent, Comparison of nacre with other ceramic composites, J. of Mater. Sci. 25, pp. 3173-3178, 1990.
[3] V. Srot, U.G.K. Wegst, U. Salzberger, C.T. Koch, K. Hahn, P. Kopold and P.A. van Aken, Microstructure, chemistry, and electronic structure of natural hybrid composites in abalone shell, Micron 48, pp. 54-64, 2013.
[4] M. Vittori, V. Srot, K. Žagar, B. Bussmann, P.A. van Aken, M. Čeh and J. Štrus, Axially aligned organic fibers and amorphous calcium phosphate form the claws of a terrestrial isopod (Crustacea), J. of Struct. Biol. 195, pp. 227-237, 2016.
[5] V. Srot, B. Bussmann, U. Salzberger, C.T. Koch and P.A. van Aken, Linking microstructure and nanochemistry in human dental tissues, Microsc. Microanal. 18, pp. 509-523, 2012.
[6] V. Srot, B. Bussmann, U. Salzberger, J. Deuschle, M. Watanabe, B. Pokorny, I. Jelenko Turinek, A. F. Mark and P.A. van Aken, Magnesium-assisted continuous growth of strongly iron-enriched incisors, ACS Nano 11 (1), pp. 239-248, 2017.
[7] H.K. Hagler, Ultramicrotomy for Biological Electron Microscopy, Methods Mol. Biol. 369, pp. 67–96, 2007.
[8] C. T. Koch, W. Sigle, R. Höschen, M. Rühle, E. Essers, G. Benner and M. Matijevic, SESAM: Exploring the Frontiers of Electron Microscopy, Microsc. Microanal. Off. J. Microsc. Soc. Am. Microbeam Anal. Soc. Microsc. Soc. Can. 12, pp. 506–514, 2006.
[9] R.F. Egerton, Electron Energy-Loss Spectroscopy in the Electron Microscope, Springer US: Boston, MA, 2011.
[10] S. Mann, Biomineralization: Principles and Concepts in Bioinorganic Materials Chemistry; Oxford University Press, 2005.
[11] H.A. Lowenstam and S. Weiner, On Biomineralization, Oxford University Press, New York, 1989.
[12] M.A. Meyers, P.-Y. Chen, A.Y.-M. Lin, Y.S eki, Biological materials: structure and mechanical properties, Progress in Materials Science. 53, pp. 1-206, 2008.
[13]S. Weiner, Organization of extracellularly mineralized tissues: a comparative study of biological crystal growth, CRC Critical Reviews in Biochemistry 20, pp. 365-408, 1986.
[14] K. Benzerara, T.H. Yoon, T. Tyliszczak, B. Constantz, A.M. Spormann, G.E. Brown Jr., Scanning transmission X-ray microscopy study of microbial calcification, Geobiology 2, pp. 249-259, 2004.
[15] T.E. Schäffer, C. Ionescu-Zanetti, R .Proksch, M.Fritz, D.A. Walters, N. Almqvist, C.M. Zaremba, A.M. Belcher, B.L. Smith, G.D. Stucky, D.E. Morse and P.K. Hansma, Does abalone nacre form by heteroepitaxial nucleation or by growth through mineral bridges?, Chemistry of Materials 9, pp. 1731-1740, 1997. 
[16] F. Song, X.H. Zhang, Y.L. Bai, Microstructure in a biointerface, Journal of Matters Science Letters 21, pp. 639-641, 2002.
[17] I.A. Mjör and I. Nordahl, The density and branching of dentinal tubules in human teeth, Archives of Oral Biology 41(5), pp. 401-412, 1996.
[18] R.Z. LeGeros, Calcium phosphates in oral biology and medicine, In Monographs in Oral Science, H.M. Myers (Ed.), Karger Basel Switzerland, 1991.
[19] E.V. Pindborg, J.J. Pindborg and C.M. Plum, Studies on incisor pigmentation in relation to liver iron and blood picture in the white rat, Acta Pharmacologica 2, pp. 285-293, 1946.
Website developed by S8080 Digital Media