Technisch-Naturwissenschaftliche Fakultät

Polarized confocal Raman microscopy as a powerful tool in the investigation of biomineralized systems

DISSERTATION

zur Erlangung des akademischen Grades

Doktor der Technischen Wissenschaften

im Doktoratsstudium der

TECHNISCHEN WISSENSCHAFTEN

Eingereicht von: DI Reisecker Christian

Angefertigt am: Institut für Polymerwissenschaften

Beurteilung: Univ. Prof. Dr. Sabine Hild

Associate Professor DI Dr. Clemens Schwarzinger

Linz, September 2015 Christian Reisecker

Diplom-Ingenieur Langwiedmoos 10 5231 Schalchen Mail: [email protected]

Persönliches geboren am 6. Jänner 1985 in Braunau am Inn Österreichischer Staatsbürger

Studium und Ausbildung seit 11/2010 Johannes Kepler Universität Linz Doktoratsstudium der Technischen Chemie Dissertations- Thema: Polarized confocal Raman microscopy as a powerful tool in the investigation of biomineralized systems Schwerpunkte: Raman Spektroskopie, Material- und Oberflächenanalytik

10/2004 – 10/2010 Johannes Kepler Universität Linz Studium der Technischen Chemie Diplomarbeits-Thema: Palladium catalyzed synthesis of novel [1, 10]-Phenanthroline derivates and controlled crystallization on Muscovite mica. Schwerpunkte: organische Synthesen, Charakterisierung mittels UV-Vis, Fluoreszenz sowie NMR-Spektroskopie

2003/2004 Abgeleistete Wehrpflicht

1999 – 2003 Oberstufenrealgymnasium in Neumarkt am Wallersee Naturwissenschaftlicher Zweig, Matura

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Weiterbildungen

5/2015 Lehrgang Projektmanagement Johannes Kepler Universität Linz Grundlagen des Projekt- und Prozessmanagements

8/2011 Lehrgang Hochschuldidaktik Johannes Kepler Universität Linz Didaktische Grundlagen, Vortrags- und Präsentationstechniken, Erstellung von Lehrunterlagen, Publikationstechniken

Vorträge und Posterpräsentation auf Fachtagungen

München, 12.12.2013 Structure, function, orientation: Investigation of biomineralized systems by means of confocal Raman microscopy. Institut für Geo- und Umweltwissenschaften, Ludwig-Maximilians Universität München

Leoben, 07.11.2013 The sea urchin tooth formation: A micro Raman study; Werkstoff-kongress 2013 Leoben

Freiberg, 28.08.2013 The sea urchin tooth formation: A polarized micro Raman study investigating the transformation from ACC to calcite and high Mg-calcite; Biomineralisations-Konferenz Freiberg (Deutschland)

Regensburg, 12.03.2013 Polarized confocal Raman microscopy and EBSD: A study outlining comparability between these methods by studying calcite crystalline regions within the tergite cuticle of terrestrial isopods; DPG-Tagung Regensburg (Deutschland)

Siofok, 28.05.2012 Selective Decalcification: A method to determine the structure within the cuticle of isopods; Biopolymer-Konferenz Siofok (Ungarn)

Graz, 23.09.2013 Poster-Präsentationen: Scanning confocal Raman microscopy as a powerful tool in the investigation of biomineralized systems; Österreichische Chemietage Graz

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Berlin, 25.03.2012 Poster-Präsentationen: Selective decalcification: A method to determine the phosphate distribution within the cuticle of isopods by confocal Raman microscopy; DPG Tagung Berlin (Deutschland)

München, 12.03.2012 Poster-Präsentationen: Structural investigation of isopod cuticle and changes caused by decalcification monitored by sub µ-confocal Raman microscopy; DGK-Konferenz München (Deutschland) Publikationen

November 2012 Bastian H.M. Seidl, Christian Reisecker, Sabine Hild, Erika Griesshaber, Andreas Ziegler; Calcite distribution and orientation in the tergite exocuticle of the isopods Porcellio scaber and Armadillidium vulgare (Oniscidea, Crustacea); Zeitschrift für Kristallographie-Crystalline Materials; Volume 227, Number 11, page 777-792.

Februar 2013 Notburga Gierlinger, Christian Reisecker, Sabine Hild, Sonja Gamsjäger; Raman microscopy: Insights into the Chemistry and Structure of Biological Materials; Chapter 7, page 151-180; Book: Materials Design Inspired by Nature; Royal society of chemistry;

April 2013 Sukhum Ruangchai, Christian Reisecker, Sabine Hild, Andreas Ziegler; The architecture of the joint head cuticle and its transition to the arthrodial membrane in the terrestrial crustacean Porcellio scaber; Journal of Structural Biology; Volume 182, Number 1, page 22-35.

August 2014 Francisca Alagboso, Christian Reisecker, Sabine Hild, Andreas Ziegler; Ultrastructure and mineral composition of the cornea cuticle in the compound eyes of a supralittoral and a marine isopod; Journal of Structural Biology; Volume 187, Number 2, page 158-173.

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Eidesstattliche Erklärung

Ich erkläre an Eides statt, dass ich die vorliegende Dissertation selbstständig und ohne fremde Hilfe verfasst, andere als die angegebenen Quellen und Hilfsmittel nicht benutzt bzw. die wörtlich oder sinngemäß entnommenen Stellen als solche kenntlich gemacht habe. Die vorliegende Dissertation ist mit dem elektronisch übermittelten Textdokument identisch.

Linz, September 2015

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Danksagung

Ich bedanke mich bei Frau Dr. Sabine Hild für die interessante Themenstellung und nicht zuletzt die umfangreiche Betreuung und Schaffung der Grundvoraussetzungen, die es mir ermöglicht haben diese Dissertation durchzuführen.

Ein großes Dankeschön an Herrn Dr. Andreas Ziegler und Sukhum Ruangchai von der Universität in Ulm, sowie Dr. Erika Griesshaber von der LMU München für die vielen Proben und spannenden Diskussionen.

Für die vielen schönen Stunden am Institut möchte ich mich bei den Kollegen Rudolf Hasslacher, Bernhard Jachs, Lisa Uiberlacker, Moritz Strobel, Martin Laher, Günther Gratzl, Tobias Keplinger, Thomas Fischinger und Susanne Kimeswenger bedanken, dass es nie langweilig geworden ist.

Danke auch an meine besten Freunde Florian Lengwin, Michael König und Lorenz Reith für die kurzweiligen Stunden mit euch Abseits des Uni-Alltags.

Ganz besonders möchte ich mich bei meinen Eltern Hermine und Josef Reisecker für ihre Unterstützung in allen Lebenslagen bedanken. Ohne eure Unterstützung wäre es nie möglich gewesen meine Ziele zu erreichen.

Am meisten möchte ich mich bei meiner Frau Antonia Reisecker bedanken. Du bist mir immer beigestanden und auch in schwierigen Stunden für mich da gewesen. Es ist ein großes Glück dass ich dich an meiner Seite habe!

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Content 1 Einleitung ...... 13 2 General aspects on confocal Raman microscopy ...... 17 2.1 History of Microscopy and Raman spectroscopy ...... 17 2.1.1 History of Microscopy ...... 17 2.1.2 History of Raman Spectroscopy ...... 18 2.2 Raman micro spectroscopy in the field of biocomposites ...... 19 2.3 Molecule vibrations and degree of freedom ...... 20 2.4 General physical background of Raman/IR and energy transfer ...... 23 2.4.1 Elastic and inelastic light scattering ...... 24 2.4.2 Quantum mechanical description of the Raman effect ...... 27 2.5 Confocal microscopy and resolution limits...... 29 2.5.1 The lateral resolution of a microscope (xy-axis) ...... 29 2.5.2 The axial resolution of a microscope (z-axis) ...... 30 2.5.3 Confocal microscope ...... 32 2.5.4 Image formation within a confocal microscope ...... 33 2.5.5 Confocal Raman microscope ...... 33 2.5.5.1 The pinhole of a confocal Raman microscope ...... 34 3 Materials and Methods ...... 34 3.1 Materials ...... 34 3.1.1 Spectral reference samples ...... 34 3.1.2 Isopod tergite-, joint head- and eye-cuticle ...... 36 3.1.3 The sea urchin tooth of Paracentrotus lividus ...... 36 3.2 Methods ...... 37 3.2.1 Confocal Raman microscopy ...... 37 3.2.2 Instrument calibration ...... 40 3.2.3 Data evaluation ...... 40 3.2.3.1 Cosmic ray removal ...... 40 3.2.3.2 Baseline correction ...... 40 3.2.3.3 Raman spectral image generation / Univariate data analysis ...... 41 3.2.3.4 Multivariate data analysis ...... 43

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3.2.4 Reference spectra and interpretation ...... 46 3.2.5 Orientational properties of calcite ...... 51

3.2.6 Mg-calcite and the determination of the MgCO3 content ...... 53 3.2.7 Polymorphs of calcium carbonate ...... 54 4 Investigation of different isopod cuticle systems ...... 57 4.1 General aspects on isopods and their cuticle ...... 57 4.2 The tergite cuticle of isopods ...... 58 4.2.1 The overall chemical composition of the tergite cuticle for several interesting isopod species ...... 61 4.3 Results and discussion...... 62 4.3.1 Determination of the local chemical distribution with confocal Raman microscopy ...... 62 4.3.2 Organic material within the tergite cuticle (epicuticle, chitin as matrix within the endocuticle and membranous layer) ...... 64 4.3.2.1 The epicuticle as outmost layer within the tergite cuticle of isopods 64 4.3.2.2 Chitin protein matrix within the endocuticle and membranous layer and its orientational properties ...... 64 4.3.3 Inorganic components within the tergite cuticle (exocuticle, ACC and phosphate within the endocuticle) ...... 73 4.3.3.1 The exocuticle of the isopod tergite cuticle...... 73 4.3.3.2 Orientational properties within the calcite crystalline region of isopod tergite cuticle systems ...... 75 4.3.3.3 Calcite orientation within the tergite cuticle of Porcellio scaber ...... 75 4.3.3.4 Calcite orientation within the tergite cuticle of Armadillidium vulgare ...... 78 4.3.3.5 Calcite orientation within the tergite cuticle of Tylos europaeus ...... 81 4.3.3.6 Calcite orientation within the tergite cuticle of Helleria brevicornis 84 4.3.3.7 The exocuticle of the marine isopod Sphaeroma serratum ...... 85 4.3.3.8 ACC and phosphate within the endocuticle of the isopod tergite cuticle...... 86 4.3.4 Deviations from classical tergite cuticle structuring / the properties of thoracomere edges ...... 89

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4.4 The joint head cuticle of Porcellio scaber, Tylos europaeus and Helleria brevicornis ...... 92 4.4.1 The joint head cuticle of Helleria brevicornis and Tylos europaeus ...... 97 4.4.2 The coxal plates of Helleria brevicornis and Tylos europaeus ...... 100 4.5 The cornea eye cuticle of Sphaeroma serratum and Ligia oceanica ...... 101 4.5.1 The eyes of Ligia oceanica ...... 102 4.5.1.1 The head capsule of Ligia oceanica ...... 102 4.5.1.2 The cornea cuticle of Ligia oceanica ...... 105 4.5.2 The eyes of Sphaeroma serratum ...... 106 4.5.2.1 The head capsule of Sphaeroma serratum ...... 106 4.5.2.2 The cornea cuticle of Sphaeroma serratum ...... 108 5 Sea urchin tooth ...... 110 5.1 The start of the tooth growth at the posterior edge (plumula) ...... 113 5.2 The center of the tooth ...... 125 5.3 The tip (anterior edge) of the sea urchin tooth ...... 128 6 Conclusion ...... 135 7 Appendix ...... 137 7.1 Average spectra of the endocuticle (tergite cuticle of several isopod specimen) ...... 137 7.2 Average spectra of the membranous layer (tergite cuticle of several isopod specimen) ...... 138 7.3 Average spectra of the exocuticle (tergite cuticle of several isopod specimen) ...... 139 7.4 CR-values for the anterior and posterior edge of Tylos europaeus ...... 139 7.5 CR-values for sagitally prepared exocuticle of Helleria brevicornis ...... 141 7.6 Average spectra of the phosphate enriched parts in the endocuticle (tergite cuticle of several isopod specimen) ...... 142 7.7 CR-values for sagitally prepared joint head cuticle of Porcellio scaber ...... 143 7.8 Average spectra of the joint head cuticle of Helleria brevicornis ...... 144 7.9 Average spectra of the joint head cuticle of Tylos europaeus ...... 145 7.10 Average spectra of selected regions within the coxal plate cuticle of Helleria brevicornis ...... 146

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7.11 Average spectra of selected regions within the coxal plate cuticle of Tylos europaeus...... 147 7.12 Average spectra of the head capsule of Sphaeroma serratum ...... 148 7.13 The average spectra according to the cluster analysis for the cornea cuticle of Sphaeroma serratum ...... 149 7.14 Average spectra of the central and edge region at cross section level 1 starting from the posterior edge of the sea urchin tooth of Paracentrotus lividus...... 150 7.15 Average spectra of section 2 and 3 within the central region of the sea urchin tooth...... 151 7.16 Average spectra according to the cluster analysis performed at level 2 at the anterior edge of the sea urchin tooth of Paracentrotus lividus...... 152 7.17 Average spectra according to the cluster analysis performed at level 3 at the anterior edge of the sea urchin tooth of Paracentrotus lividus...... 153 7.18 Average spectra according to the cluster analysis performed at the anterior edge of the sea urchin tooth of Paracentrotus lividus in tangential fashion...... 154 8 List of Figures ...... 155 9 List of tables ...... 169

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Abbreviation

ACC amorphous calcium carbonate

ACP amorphous calcium phosphate

AFM atomic force microscopy app. approximately br. broad

BSA bovine serum albumin cc cornea cuticle

CCD charge coupled device cpp. carinar process plates

CR carbonate ratio cts. counts

EBSD electron backscatter diffraction

EDX energy dispersive X-Ray spectroscopy

FWHM full width at half maximum

HA hydroxyapatite hc head capsule ms milli seconds mW milli watt nm nanometer

PCA Principal component analysis pp primary plates

PSF point spread function

SEM scanning electron microscopy

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SERS surface enhanced Raman scattering sp secondary plates str. vib. stretching vibration

TEM transmission electron microscopy

TERS tip enhanced Raman scattering wd working distance

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1 Einleitung

Die konfokale Raman Mikroskopie und ihre Anwendung in der Wissenschaft hat in den letzten Jahrzehnten immer mehr an Bedeutung gewonnen. Einer der wesentlichen Gründe hierfür sind die Entwicklungen auf technischer Ebene wie zum Beispiel die stetige Verbesserung von CCD Detektoren, die Integrationszeiten von unter 50 ms erlauben und trotzdem hoch aufgelöste Spektren liefern. Die Kombination eines Raman Spektrometers mit einem konfokalen Mikroskop jedoch war einer der wesentlichen Gründe warum sich Raman Mikroskopie immer größerer Beliebtheit erfreut, da hierbei räumliche Auflösungsgrenzen von bis zu 400 nm erzielt werden können. Die Anwendung von Raman Mikroskopie auf Biokomposit Ebene bringt wesentliche Vorteile mit sich, da zum Einen unterschiedlichste anorganische und organische Materialien unterschieden werden können, zum anderen aber auch kristalline Polymorphe anhand der vom Kristallgitter verursachten Gitterschwingung wie zum Beispiel Calcit, Aragonit und Vaterit unterschieden werden können. Durch die Anwendung von Laser Licht mit einer definierten Polarisationsrichtung können zudem auch orientierungsabhängige Eigenschaften, wie zum Beispiel Chitin Faserorientierung oder Calcit Ausrichtung bestimmt werden. Durch das Erstellen einer sogenannten Raman map können ganze Flächen abgerastert werden und so die örtliche Zusammensetzung von Proben unterschiedlichster Art bestimmt werden. Die Anwendung von Raman Mikroskopie auf Isopoden Ebene ist sehr nützlich, da die Isopoden Kutikula ein gutes Rollenmodell für biomimetische Anwendungen darstellt, weil diese Kutikula unterschiedlichste Eigenschaften wie Härte an der Außenseite (zur Abwehr von Fressfeinden), aber auch eine weiche Innenseite, die es erlaubt das Muskeln anbinden in sich vereint. Dies geschieht durch die Anwendung unterschiedlichster Materialien wie zum Beispiel Calcit, amorphes Calciumcarbonat, einer Chitin Protein-Matrix, Phosphaten und diversen anderen organischen Materialien unter Einhaltung einer strikten hierarchischen Bauweise auf unterschiedlichsten Größenebenen. Je nachdem welchen Verteidigungsmechanismus die jeweilige Spezies verfolgt, wird die Konstruktion in Punkto Dicke und Zusammensetzung nach den Erfordernissen angepasst. Hierbei zeigt es sich, dass auch der Lebensraum einen großen Einfluss auf die Zusammensetzung der Kutikula ausübt. Durch den Einbau von

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Magnesium in das Calcit Kristallgitter kann außerdem auch noch die Härte der Außen Schicht modifiziert werden. Der Aufbau der Chitin Protein Matrix ist äußert Komplex, da selbige aus 7 hierarchischen Ebenen besteht und nach außen hin wie eine verdrehte Helix aussieht. Selbige wird durch den zusätzlichen Einbau von amorphem Calciumcarbonat verstärkt. Neben der Kutikula sind auch Gelenke und Augen der Isopoden von Interesse für biomimetische Zwecke, da erstere einen ähnlichen Aufbau wie die Kutikula aufweisen, jedoch an bestimmten Stellen, wo zum Beispiel höhere Kräfte wirken, Modifikationen auftreten um ebendiesen bestmöglich entgegenzutreten. Dies ist auch von großem Interesse für medizinische Anwendungen, da ein Gelenk aus Biokompositen so gut wie keine Abstoßungsreaktionen zeigen würde, weil die meisten Komponenten ohnehin bereits im Körper vorhanden sind. Die Untersuchung der Facettenaugen liefert viel neues Wissen zur Funktion und Wirkung ebendieser und wie auch diese von Art zu Art in punkto Form und Anzahl und Struktur der Facetten variiert. Die Untersuchung des Seeigelzahns ist von großem Interesse, da selbiger bedingt durch den Lebensraum am steinigen Meeresgrund und der damit verbundenen Abnützungserscheinungen kontinuierlich erneuert werden muss. Der Zahn selbst besteht zum größten Teil aus Mg2+ angereicherten Calcit. Ein vollständiges Seeigelzahn Gebiss besteht aus 5 Zähnen, welche über den Kieferknochen fixiert und ausgerichtet sind. Dabei bildet der Zahn 5 unterschiedlich orientierte Bereiche aus. Es handelt sich hierbei um die sogenannten Primär- (pp) und Sekundärplatten (sp), den Stein, die Prismen sowie die carinar process Platten (cpp). Das Wachstum geschieht am Ende des Zahnes in der sogenannten “Plumula“. Aus amorphem Calciumcarbonat werden erste Calcit Platten geformt. Ab einer gewissen Anzahl dieser Primärplatten werden die Sekundärplatten und der Stein gebildet. Am Schluss werden die Calcit Fasern gebildet, welche stetig an Durchmesser zunehmen und dann den Bereich der Prismen bilden. An der Aussenseite dieses Prismen Bereichs befinden sich dann die carinar process Platten. Über die gesamte Dauer der Zahnbildung wird außerdem der Mg2+ Gehalt sukzessive erhöht. Die höchste Mg2+ Konzentration kann im sogenannten Stein nachgewiesen werden, welcher dadurch der härteste Bereich im Seeigelzahn Konstrukt wird. Somit ist es auch logisch, dass dies der Bereich ist der an der Zahnspitze am längsten den rauen Umgebungs Bedingungen am Meeresgrund standhalten kann.

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1 Introduction Biocomposites have gained lots of attention over the last years, as they are made out of materials nature provides. Several materials are combined forming different structures, which yield outstanding physical properties in combination. These materials are of particular interest in the case of biomimetic, as quite diverse mechanical properties like hardness and softness are combined on nanometer scale within a single workpiece. The generation of biomimetic elements is also very interesting in the case of medical applications in future, as for instance knee joints formed out of biocomposites would lead to less or even no rejection reactions of the human body, as the applied materials are present within the human body anyway. But it is still a long journey to reach this goal, as science still is in the stage of understanding the connections between structure, function, orientation and composition of biocomposites provided by nature. Therefore, it is inevitable to create more knowledge on biocomposites before further biomimetic steps even on laboratory scale can be considered. Several methods for the investigation of the chemical composition in biocomposites like EDX, X-ray or EBSD and on the other hand there are microscope techniques like SEM or TEM for structural investigations are used, but none of them provides both, structural and compositional information in a single method. Confocal Raman microscopy allows a very detailed insight into the framework of biocomposites on structural and compositional level ranging from the meso- to the nano-scale. Several inorganic and organic components can be identified due to their characteristic Raman spectra, but also structural alterations like a change of the chitin fiber direction, as well as changes within the orientation of crystals can be identified with this method. In terms of minerals, polymorphic materials, which are characterized having the same chemical composition, but different Bravais lattice, can be discriminated. A good example is calcium carbonate, which can have amorphous character, and three crystalline forms, which are calcite, vaterite and aragonite. But these calcium carbonate polymorphs rarely can be found in pure form in nature as the mechanical properties can be modified via an incorporation of different ions as Mg2+ into the Bravais lattice in example. In the case of calcite, an incorporation of magnesium leads to a higher hardness of the material, which is necessary for the defense mechanism of some species forming hard shells or in the

15 protection of sensitive organs like the eye. The main advantage of confocal Raman microscopy is its high lateral spatial resolution around 400 nm depending on the microscope objective and laser wavelength. In combination with a high throughput CCD camera, integration times down to 50 ms can be achieved. The provided gratings (600 and 1800 grooves per mm) in the spectrometer ensure a high spectral resolution of 3 cm-1 and 0.9 cm-1. Therefore, high detailed information on the specimen can be generated if Raman spectral mapping is applied in a proper way. Data evaluation is another very important part, as interesting sample features like peak widths, peak center positions determined via Gauss fits or a proper performed Cluster analysis often reveals hidden features of the measured sample. Several biocomposites were investigated. Examples herein are the tergite cuticle, the joint head cuticle and the cornea cuticle of different isopod species. A great diversity on compositional, structural and orientational properties could be observed. The defense mechanism of the isopod plays an important role on these properties. The tergite cuticle in example combines quite diverse properties like hardness and softness within a single sample. The uppermost layer works as a protective shield against enemies, but the inner part of the cuticle is soft in order to enable the attachment of muscles. Pore canals for materials transport can be found within the framework and the whole cuticle also works as a barrier against water loss. Several materials like calcite, amorphous calcium carbonate (ACC), chitin and some phosphates are used to form this very complex structured cuticle system. The hierarchical structure in the center of sagittal prepared tergite samples is quite similar, but at the edges deviations from those classical structures can be observed as different mechanical requirements apply to these parts. A second very interesting role model is the sea urchin tooth of Paracentrotus lividus, as tooth material is produced continuously during its lifetime. This is necessary as the species lives on the sea ground and the teeth grind off as the species grazes around on the hard sea floor substrate. The tooth is composed of different Mg2+-enriched calcite regions as well as calcite forming several structural elements in order to meet the mechanical requirements. First, calcite primary plates (pp) are formed, followed by the incorporation of Mg2+ during maturation and forming several other structural elements like secondary plates (sp), the stone, prisms and the carinar process plates (cpp) giving the sea urchin tooth its unique T-like shape.

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2 General aspects on confocal Raman microscopy 2.1 History of Microscopy and Raman spectroscopy

2.1.1 History of Microscopy Confocal Raman microscopy is a combination of two methods. On the one hand there is a confocal microscope generating high magnifications and spatial resolution of a certain sample. And on the other hand a Raman spectrometer is attached to the microscope in order to perform Raman spectroscopy. Both techniques have a long history and therefore a brief historical review in the development of both methods is worth to look at. [1] Microscopy of course has a far greater history, as the first optical devices and lenses date back about 2000 years. The first documented microscope was manufactured by Galileo Galilei in 1609 via a combination of a convex and concave lens. Slow developments were made until the 19th century. This changed as pioneers such as Ernst Abbe investigated the physical processes in optical image formation. In combination with Otto Schott, who was an expert in manufacturing glass exhibiting different optical characteristics, microscope objectives were constructed as they are partly still state of the art today. A big progress was made during the 20th century as some new methods like fluorescence microscopy, phase contrast and dark field illumination were invented. A milestone in the field of microscopy was the invention of the confocal microscope by Marvin Minsky in 1957. The big advantage compared to conventional microscopy is the fact that only the light within the focal plane gets detected due to the geometry of the microscope and the equipped pinhole, which light has to pass in order to reach the detector. The light above or below the focal plane is restricted by the pinhole and will not reach the detector. The lateral resolution therefore is increased by a factor of 1.4, which comes along with a light loss of 95% due to the small pinhole size. As confocal microscopes are just looking at the focal plane, they are used for applications, where high depth resolution is needed. More details on functionality of a confocal microscope can be found in section 2.5.3. [1]

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2.1.2 History of Raman Spectroscopy Theoretical considerations on inelastic scattering of light by matter were made by Brillouin and Smekal in 1922. First experimental efforts on inelastic light scattering were performed by Raman and Krishnan, which date back to 1928. A filtered beam of sunlight was used as a light source, which additionally was focused on a sample. The first detector was the human eye. Further theoretical investigations were made by Cabanes, Landsberg, Mandelstram and Rocard. The first use of laser excitation dates back to 1962 (Porto and Wood) and 1963 (Stoicheff). With the application of lasers as light source, further upgrades increasing the quality of Raman spectroscopy such as holographic gratings, detectors (i.e. multi-element arrays) were invented. Fourier transformation for data procession caused further attention on Raman spectroscopy as it enables the suppression of fluorescence caused by the sample itself. In the 70´s of the 20th century first Raman micro spectrometer systems were invented, which apart from single point measurements, also allowed first rudimental Raman spectral mapping. In the late 90´s of the 20th century Raman spectroscopy got more into focus of the scientific community, as first Raman spectrometers with high resolution confocal microscopes were combined. This opened the field for high resolution Raman mapping in several axis of the sample (xyz-mapping). [2] Furthermore, combinations of confocal Raman microscopes with various other techniques like AFM (atomic force microscopy) enlarged the field of applications for Raman microscopy, as the combination of these two techniques allows to determine physical properties, such as hardness by AFM in example, but also allows the determination of the chemical composition via a Raman measurement at the same spot on the sample. Recently, a new technique for samples having a difficult topography has reached the market. A topographic sensor determines the topography of the sample, generating a z-profile. The knowledge of the z-profile is used to move the microscope part of the Raman instrument in the correct z-position having always the correct z-focus on the surface during the whole Raman mapping procedure. The urge to look at samples in even smaller dimensions as conventional Raman microscopes led to idea to combine a Raman microscope with an electron microscope, which entered the market 2014. [1]

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Several other methods to enhance the Raman effect are used nowadays. One good example is SERS (Surface Enhanced Raman Spectroscopy), where the Raman effect of the sample itself gets enhanced as the sample is placed on a noble metal surface (i.e. Au or Ag). The enhancement of the Raman signal is due to an increase of the electric field provided by the noble metal surface. SERS is often used in the detection of biomolecules as it is the case for body fluids in example. These molecules usually have a very low Raman signal and by the aid of SERS an enhancement of a factor 104-106 can be achieved. [3] TERS (Tip Enhanced Raman Spectroscopy) uses the same effect as in SERS but there is no surface coating of noble metal, but more less an AFM-cantilever tip which is coated with a noble metal. As AFM-cantilever tips are having a size around 100 nm, the Raman signal just will be enhanced at the actual position of the cantilever tip and therefore also the Raman effect can achieve a spatial resolution around 100 nm and therefore below the diffraction limit of the microscope. [4] But not only microscope techniques in combination with Raman are of particular interest. In polymer science, dynamic processes are of particular interest like a shearing process in a rheometer. But there is also curiousness on the kinetics and the chemical changes during the process. This led to the idea to combine a rheometer with a Raman microscope. The goal herein is to observe the curing reaction online with Raman microscopy during a shearing experiment. The combination sounds very promising and first successful experiments could be performed so far at the JKU in Linz.

2.2 Raman micro spectroscopy in the field of biocomposites

A short summary of former works and important scientists inspiring this work shall be given now. First there was curiosity how bacteria and various kinds of other species are able to utilize materials provided by nature in order to construct hard or soft tissue for their own use and the whole processes behind the formation of these materials. These investigations date back to the late 1980´s and pioneers in this field were Steve Weiner and Heinz Lowenstam ([5-9]). The group of Steve Weiner was first in the investigation of the formation of ACC and its transition to calcite via measurement of Raman spectra

([10]). A further important step was the investigation of CaCO3 and the resulting Raman peak shifts caused if Ca2+ is replaced by different cations ([11]). Bischoff et al.

19 investigated the influence of Mg2+ incorporation into calcite and how the Raman spectra are changing due to that incorporation ([12]). More important work in the case of biocomposites was made by Ziegler Andreas, Sabine Hild and Helge Fabritius [13-21], who were the pioneers in the field of isopods and made a huge progress in the investigation of the properties of different isopod cuticle systems. As an example, the discovery of the molting cycle of isopods due to their growth shall be mentioned [21]. Altogether a larger focus was on the investigation of the isopod properties with electron microscopy, elemental analysis, AFM and some Raman spectroscopy. Biomineralization is also an important issue for the formation of the sea urchin tooth as new tooth material is formed continuously. The two most important contributors are Artur Veis, who first made investigations concerning the sea urchin tooth in 1986 and Stuart R. Stock, who also used Raman spectroscopy for their investigations. [22-23] These works were all of great importance as a starting point for this work.

2.3 Molecule vibrations and degree of freedom

As far as geometry of a molecule is concerned, each molecule can be characterized via several types of symmetry elements. This can either be the identity element E, which just means a rotation of 360° around the molecule axis. Another symmetry element is the rotational axis Cn, where n represents the number of necessary rotations in order to reach the starting position of the molecule. Further elements are symmetry planes either in parallel (σv) or perpendicular (σh) fashion. Another possibility is the element of inversion (i) and a combination of rotation an inversion named (Sn). Molecules having the same symmetry elements are combined within a distinct point group. Knowing the symmetry of a molecule makes it way easier to estimate the number of vibrations. Each molecule is able to perform several types of movements depending on its structure and energy even without any change of the overall energy. The whole energy a molecule in its ground state possesses can be divided in degrees of freedom describing the different movement types. Each molecule has 3n degrees of freedom, where n means the total number of atoms the molecule is composed of. Already three degrees of freedom are used to describe the translational movement of the molecule. Another three are taken to describe the rotational movement of the molecule in space. Considering this type of

20 movements, each molecule has 3n-6 degrees of freedom left for further vibrational movement. Linear molecules are excluded from this rule as they just have 2 rotational movement possibilities. Therefore, for linear molecules the 3n-5 rule applies. Oxygen as an example will have just one degree of freedom, as the degree of freedom rule for linear molecules proposes, which will be a stretching vibration along the molecule axis. Water as another example therefore has 3x(3) -6= 3 degrees of freedom. These 3 left degrees of freedom can either be an in-phase and out-of-phase stretch and deformation vibration as shown in Figure 1A-C. For linear molecules there are just 2 possible rotational vibrations yielding 3n -5 degrees of freedom. Linear molecules like CO2 therefore have 3x(3) -5= 4 degrees of freedom. In addition to the rotational and translational vibrations, there is an in-phase and out-of-phase stretching vibration as well as two mutually perpendicular deformation or bending vibrations, which are depicted in Figure 1D-G. [24, 25]

Figure 1: A-C) The three degrees of freedom for a nonlinear molecule (i.e.) water; D-G) degrees of freedom for a linear molecule (CO2); A, D are symmetrical stretching movements, B, E and F bending or deformation and C, G are asymmetric stretching movements.

It is quite obvious that the number of possible vibrations is increasing quite fast with the number of atoms forming a molecule. A classical method to explain the molecule movements is the spring and ball model. The molecule bond in this model is representing the spring and the atoms are simulated with balls. Increasing mass of atoms as well as lower bond strength come alongside with a lower frequency of the corresponding bond and vice versa but will be described more detailed later on. [24] The electronic states of a molecule can be described by the Morse curve, as depicted in Figure 2, outlined as a curved line. Within this illustration, the energy of the system is plotted against the difference of the distance between the atoms. The lowest energy level that atoms can reach is the ground electronic level, which is exactly at the bond

21 length of the participating atoms (ν=0). This is the critical distance, where the attractive energy in order to form a bond changes into repulsive nuclear forces. On the one hand, if the correct amount of energy is absorbed and the first level is reached, the molecule starts vibrating, but on the other hand if the amount of energy is higher than vmax, the atoms of the molecule are free. The distances between the various vibrational levels are not the same, which means that different amounts of energies are needed to reach several levels. Overtone vibrations can occur, if there are multiple amounts of energy reaching a certain vibrational level. Generally these type of vibrations are very weak or cannot be detected either. Another possibility is the combination of different vibrations which have the same energy, named combination bands. The diagram in total would be even more complex if rotational levels were shown, which are usually at lower energies as vibrational levels. [24]

nuclear separation distance r

Figure 2: The Morse curve illustrating the vibrational levels of an electronic state. The plot shows the typical vibrations having energy on the y-scale and the distance r between the two atoms on the x-scale.

As the Morse curve is difficult to handle for the calculation of the vibrational energies, it is replaced by the previously mentioned ball and spring model. Herein, the energy curve is represented by a parabola. The Hook´s law can be utilized to determine the frequency of a certain bond as shown in equation (1). In this equation the velocity of light in vacuum (2.99*108 ms-1) is represented by c, π is the mathematical constant expressing the ratio of a circle´s circumference to its diameter (3.14159). K is the force constant of the bond and μ is representing the reduced mass of the involved atoms, which can be calculated according to equation (2). [24]

22

1 K   (1) 2c 

M M   A B (2) M A  M B

With that knowledge it is easy to determine the frequency of the interesting bond. The force constant of course differs for a single- (3-6 millidynes/Angstrom), double- (10-12 md/Å) and triple-bond (15-18 md/Å). One millidyne is equal to 10-8 N. Except of the character of the bond, it can be stated that the lower the weight of the participating atoms, the higher the frequency of the vibration will be and vice versa. C-H vibrations for instance can be detected around 3000 cm-1 depending on the character of the whole molecule, and the C-Cl vibration will be somewhere between 560-830 cm-1.[24, 25]

2.4 General physical background of Raman/IR and energy transfer

When light (in form of photons) interacts with matter, it can either be absorbed, scattered or it just passes without any interaction. Light itself can be described as electromagnetic radiation consisting of a sinusoidal wave like motion of electric and magnetic fields. For Raman and IR spectroscopy only the magnetic field is of importance. The goal of Raman and IR is the investigation between the interaction of radiation and the energy states of the interesting molecule. Therefore, a connection between energy E and the sinusoidal wave character of light is necessary. Several physical quantities like the wavelength λ (length of a single wave [nm]), the frequency ν (number of cycles per time) and frequency or wavenumber v are needed, which are connected according to eq. (3)-(5) . In these formulas h stands for Planck´s constant (6.626*10-34 Js) and c is the velocity of light in vacuum (2.99*108 ms-1). [24, 25]

E  h (3) c   (4)   1    (5) c 

23

According to eq.(3)-(5), the energy therefore is proportional to the reciprocal wavelength. This leads to the conclusion, the shorter the wavenumber, the higher the energy and vice versa. Two examples on the opposite sides of the electromagnetic spectrum shall illustrate this fact. Gamma X-Rays are known to have a very short wavelength of 10-11 m, which leads to an energy of 1.97*10-15 Js. Microwaves on the contrary have a wavelength around 10 m yielding an energy of 1.97*10-24 Js. [25] Comparing Raman and IR-spectroscopy the way of excitation is different. In the case of IR-spectroscopy, the radiation is covering a broad range of frequencies applied on the sample. Having an energy match between the incident radiation and an energy gap between two energy levels, absorption of energy occurs and the molecule gets promoted to a vibrational excited state. The excitation step of course consumes energy and the difference in energy before and after the absorption process can be determined. Raman on the other side uses a single frequency of radiation to excite the sample. But only a very small part of the light is scattered by the molecule. Scattering also occurs if there is no energy match between the light and the molecule. Possible events are elastic (no change in the energy) or inelastic (either loss or gain of energy) scattering events and will be explained more detailed in the next section. [24, 25]

2.4.1 Elastic and inelastic light scattering All light scattering events can be described as an emission of electromagnetic radiation, which are having its origin in oscillating dipoles induced by the molecule via the electromagnetic field of the incident radiation. [24] An overview of the energy states and the possible energy transfer effects are illustrated in Figure 3. In the case of the light scattering, three possible events can take place. Rayleigh scattering, Stokes Raman scattering and Anti Stokes Raman scattering are all two photon processes. First, a photon originating from the incident light is absorbed, yielding in a transition from the electronic ground state into a virtual energy state, where a new photon is released and scattered from the virtual energy state. The virtual energy state differs in each Raman system as it depends also on the energy and the wavelength of the applied laser. [24] The most probable scattering event after reaching the virtually energy state is named Rayleigh scattering. In this case no energy is lost, because it is a totally elastic scattering

24 process. The electron cloud relaxes without any nuclear movement. Much more unlikely are events, in which the photon is scattered inelasctically (1 out of 106-108 photons). When light and electrons interact and the nuclei starts to move at the same time this event can happen. As the nuclei are heavier than electrons, a change in the energy to higher or lower energy levels can be noticed after the scattering process. If the scattered photon has less energy compared to the incident photon “Stokes scattering” is taking place. On the contrary if the released photon has more energy compared to the incident photon, “Anti stokes scattering” is happening, which is an even more uncommon event than Stokes scattering. Therefore, Anti stokes scattering is showing less intensity compared to Stokes scattering. The temperature within the system is an important parameter. With increasing temperature, molecules will more likely have a higher ground vibrational state and therefore the total amount of Anti stokes scattering will increase. As mentioned previously the energy of the virtual energy state is determined by the wavelength of the incident laser. The Boltzmann equation (equ.(6)) can be utilized to calculate the ratio of molecules in their ground vibrational and excited vibrational levels and therefore is a good estimation of the ratio between Stokes and Anti stokes scattering. [24]

(En Em ) N g   (6) n  n e kT  Nm gm

Nn and Nm are the numbers of molecules in the excited (n) and ground (m) vibrational energy level. The degeneracy is represented by g and (En-Em) is the energy difference between those vibrational energy levels. The symbol for the temperature is T and the Boltzmann constant is represented by k (1.3807*10-23 JK-1). [24]

Vibrations can occur in several ways, but the energy can be the same and therefore there is no possibility to identify them separately if 2 different components are having the same vibration at equal peak position. The total amount of these components is named degeneracy g. For most states g will be equal to 1, but as all possible vibrational states have to be considered, g can also have 2 or 3 as value for degenerate vibrations. [24]

25

excited electronic state

virtual energy state

ground electronic state

IR Anti- Rayleigh Stokes- Fluorescence stokes scattering scattering

Figure 3: Diagram of possible energy transfer effects. The lowest energy level is at the bottom of the ground electronic state. For IR, absorption of light in a higher level of the ground electronic place takes place. For the Raman effect, much more energy is needed to reach the virtual energy states. For Rayleigh scattering no change in the total energy takes place. For Anti stokes and Stokes scattering either a gain or loss of energy after the scattering process in total takes place. Fluorescence requires even more energy in order to reach the electronic excited state.

Stokes and Anti Stokes scattering can be shown more clarified via a plot of the wavenumber (cm-1) (of the scattered photons) versus the intensity (cts.) of the photons. The Rayleigh peak is centered at 0 wavenumbers by definition as illustrated in Figure 4, because no energy change is taking place due to the complete elastic scattering. In these spectra, each peak corresponds to a certain vibration or a combination of vibrations of the investigated system. Stokes scattering is plotted on positive wavenumbers, whereas Anti Stokes are found on the negative wavenumber side of the spectrum. Anti Stokes additionally show lower peak intensity as Anti Stokes scattering is less common than Stokes scattering. As the Raman effect itself represents a rare event, and due to the intensity differences of Stokes and Anti Stokes, predominantly Stokes scattering is investigated in the Raman scientific community. [24, 25]

26

Rayleigh peak

Stokes side of the spectrum

Anti Stokes side of the spectrum

-400 -300 -200 -100 0 100 200 300 400 wavenumber / cm-1

Figure 4: Classical Raman spectrum of calcite showing the Rayleigh peak, Stokes and Anti Stokes peaks. As Anti Stokes scattering is less common to Stokes scattering, the peak intensities are weaker.

2.4.2 Quantum mechanical description of the Raman effect A more mathematical but necessary description of the Raman effect shall be given now. The charge distribution of the molecule gets disturbed by the primary incident electric field E inducing a dipole moment μ according to equation (7). This is also the case for nonpolar systems. The total amount of the dipole moment acts as a macroscopic polarization. As the polarizability α, which is the ability of electrons to polarize, differs for each molecule, it has also been taken into account in equation (7). This is the source for the molecule generating a secondary field, which is the scattered light. [1, 24, 25]

 E (7)

The real part of electric field E can be described by its vector amplitude E0 and the oscillation frequency ω0 as depicted in equation (8). [1, 24, 25]

27

E  E0 cos(0t) (8)

As the system tries to minimize the energy, nuclear motion changes the polarizability due to adjustment to the momentarily nuclear geometry. For a description of this effect the polarizability is transformed into a Taylor series (equation (9) q  q0 cos(qt) )

3 around the equilibrium nuclear geometry (Q0). All the individual modes q are described by Q. More details are described in literature [24, 25]. Oscillations having the characteristic frequency ωq along the normal coordinate q have to be established in order to solve the Taylor series as shown in equation (10). [1]

N    1  2     (Q)      q   qq´Oq3 (9) 0  q  2  qq´ q1     (10)

The next step is inserting equation (8) and (9) into (7). The sum over all modes is reduced to a mode q and the Taylor series simplified assuming an electrical harmonic approximation. Solving the trigonometric functions yields the following formula describing the dipole moment within a molecule. [1]

Rayleigh scattering Stokes scattering

1    (t)  0  E0 cos(0t )     q0  E0 cos(0 q )t 2  q qo (11) 1        q0  E0 cos(0  q )t 2  q qo Anti-Stokes scattering

Equation (11) can be divided in three separate frequency terms forming the time dependent induced dipole moment, which is responsible as energy source for the scattered radiation. As there is no change within the frequency in the first term compared to the incident radiation, this is the completely elastically scattered part of the radiation, thus Rayleigh scattering. In the second and third term, there are differences in

28 the frequency between the laser and the normal mode frequency. The second term is representing Stokes and the third Anti-stokes scattering as highlighted in equation (11). Selection rules decide whether a vibration is Raman or IR active. The geometry of the molecule is an important factor, which also has to be taken into account. For Raman activity, a change in the polarizability is necessary, whereas for IR a change in the dipole moment is required. Symmetric systems (i.e. symmetric stretching vibration) do not have a permanent dipole due to the opposite direction of the induced dipole moment yielding a value of zero. The change in polarizability refers to the electron density within a molecule. Increasing inter-nuclear distances come alongside with an increase in polarizability. Antisymmetric stretching vibrations of a triatomic molecule for instance is an example for an IR-active vibration, as the sign of the dipole moment changes going through the equilibrium configuration. [1, 24, 25]

Fluorescence, which often causes problems in Raman spectroscopy, shall also be discussed briefly. First of all, an absorption process of a photon is necessary moving the electron to an excited state. If it is not the lowest level within the excited electron state, non-radiative transition to the ground level takes place. Moving downwards to the ground electron state radiation is emitted, which is named fluorescence. Fluorescence often means a problem in Raman spectroscopy as it masks the characteristic vibrations of the investigated molecules via a dominant base line and therefore a detailed characterization is nearly impossible. [26]

2.5 Confocal microscopy and resolution limits

2.5.1 The lateral resolution of a microscope (xy-axis) The general history of microscopy was already discussed in section 2.1.1. But now, a more detailed insight into microscopy itself and important limitations shall be given. The optical or lateral resolution can be determined via the diffraction of light by the sample and the objective lens. The lateral resolution therefore determines the lowest possible distance between two points, where still discrimination is possible with the applied setting without performing oversampling. If the limit of resolution applies point objects, the object point is not infinitely small, but can be described as a circular point. [27] In the case of confocal Raman microscopy, the point size, which is equal to the lateral resolution (rmin), is determined via the laser spot size, which depends on the

29 excitation wavelength of the laser (λ0), as well as the numerical aperture (NAobj) of the used objective. The relationship therefore is described by the Abbe equation as follows in equation (12). [27]

0.61 0 rmin  (12) NAobj

Generally the numerical aperture is a dimensionless number determining the angles, allowing the individual system (microscope objective) emit or collect light, where additionally the medium between the system and sample plays and important role. The numerical aperture is defined as the product of the sin (of the half angle of the cone of light from the specimen plane accepted by the objective) and the refractive index (nd) of the medium between the sample and the objective as illustrated in equation (13). [27]

d NAobj  n sin() (13)

Therefore, the lateral resolution varies in quite big ranges depending on the medium between the sample and the objective (for a certain light wavelength). Generally it can be stated that objectives working with media having a higher refractive index, also have a higher lateral resolution. An overview of the available objectives with the corresponding lateral resolution can be looked up in Table 2. Furthermore, the working distance (wd) is listed, which is very important in the handling of microscope objectives. The working distance is the necessary distance between the objective and the sample in order to focus the sample. [27]

2.5.2 The axial resolution of a microscope (z-axis) The axial resolution is determined along the axis of the microscope, thus perpendicular to the xy-plane where the lateral resolution was determined. A 3D diffraction image of a point source, which is formed near the focal plane, is utilized to describe axial resolution. Therefore, the axial resolution (zmin) is defined as the minimum distance between two diffraction images of corresponding points can approach to each other along the z-axis and still be distinguished. λ0 again represents the wavelength of the excitation source, nd the refractive index of the medium between the surface of the sample and the objective and NAobj the numerical aperture of the objective. [27]

30

2 nd z  0 min 2 (14) (NAobj ) For the determination of the axial resolution of the different microscope objectives, knowledge of the refractive indices of the applied measuring media is necessary. An overview of the most important media shall be given in Table 1. [28]

Table 1: Available media and their correspondent refractive index nd. [28]

medium refractive index nd

air 1

water immersion 1.33

oil immersion 1.52

Considering the refractive index of the different media it is very simple to determine the axial resolution for the microscope objectives, which is shown in Table 2.

Table 2: Available microscope objectives with different magnification and their numerical aperture, working distance and the calculated lateral (rmin) and axial (zmin) resolution determined according to equation (12) and (14).

numerical working objective type aperture distance rmin / nm zmin / µm NA wd/ mm

Zeiss Epiplan 20x 0.4 3.1 810 6.65

Nikon 50x 0.8 1 405 1.66

Nikon 100x 0.9 0.26 360 1.31

Nikon NIR-APO 60x 1 2 325 1.42 water

Nikon achromat 60x 0.8 0.25 405 2.53 air/oil

Nikon 100x oil 1.25 0.23 260 1.04

31

2.5.3 Confocal microscope In terms of a Zeiss microscope a white light or monochromatic light source passes a beam splitter and is focused via an objective at the sample as shown in Figure 5. As the microscope works in backscattered geometry, the light again passes the objective and gets focused onto the pinhole, which is the most important part in a confocal microscope. Beyond the pinhole, a camera or detector is located. [1, 24, 25] The pinhole has a certain diameter, which determines the amount of light passing. In case of the used system, three different pinhole diameters can be applied, which are 100 µm, 50 µm and 25 µm. The smaller the diameter of the pinhole, the lower the amount of light passing through it. The big advantage of confocal microscopes is the fact that due to the geometry, just the light interacting with the sample in the focal plane is allowed to pass the pinhole, which means a big enhancement in image contrast. All other light at different planes is restricted in passing the pinhole as depicted in Figure 5 (highlighted as reference plane). The selection of a proper pinhole size can increase the lateral resolution by a factor of up to 2 . Another advantage of a confocal microscope is the fact that only a small spot is measured, which enables measurements of a sample surface in xy and even z direction, generating an overview over big samples. [1, 24, 25]

beam of focal plane pinhole

beam of reference plane

point-like light/laser source beam splitter

objective

focal plane reference plane Figure 5: Scheme of a confocal microscope with a point-like light source. Just the light interacting with the sample in the focal plane is allowed to pass the pinhole, the rest is restricted (reference plane).

32

2.5.4 Image formation within a confocal microscope The point spread function (PSF) is one of the most important properties of a confocal system, which differs for each microscope objective. The PSF describes how an idealized point like object is reproduced by the underlying system. In terms of microcopy bending effects are limiting the PSF and therefore are also limiting the resolution limit of the used objective. The PSF can either be electrical, magnetic or the total energy density in the focus. For the detection, the PSF can be different depending on the emitted light and the detector. Determining the PSF yields information not only on the amplitude of the function but also the phase, which is important information in coherent image formation. As a confocal microscope applies point like excitation and detection, the image formation can be described as coherent. Comparing a conventional with a confocal microscope the full width at half maximum (FWHM) of the intensity

PSF decreases by a factor of 2 . [1, 29]

2.5.5 Confocal Raman microscope Due to the nature of Raman scattering, the image formation differs compared to a normal confocal microscope. The intensity distribution for a confocal Raman microscope is described in equation (15)-(17). As the intensities of excitation (h1) and scattering (h2) are the same in the case of confocal Raman microscopy, the intensity is to the square (heff). The expression f represents the Raman generation in x, y and z direction. The optical coordinates u and v refer to the incident excitation light having the wavelength λ1 as shown in equation (16) and (17). λ2 is representing the scattering light wavelength but is more like an assumption, as the scattered light wavelength is more less a small distribution of different wavelengths. [1, 24, 25]

2 (15) I  heff  f  u v  heff  h1u,vh2 ,      (16)

   1 (17) 2

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2.5.5.1 The pinhole of a confocal Raman microscope Generally, the size of the pinhole can be adjusted in two distinct ways depending on the chosen set up. On the one hand there is the possibility to adjust the pinhole manually or automatically via micrometer screws. And on the other hand there are systems, in which the pinhole size is determined via the diameter of an attached multi-mode fiber. The size of the pinhole has to be chosen carefully, as a smaller pinhole means higher resolution but also a loss in signal intensity and vice versa. Therefore, an estimation between these two parameters has to be made in order to receive the best results. A relation between the objective parameters and the pinhole as well as the laser parameters is helpful to estimate the necessary pinhole size and is mentioned above in equation (18). [1, 24, 25] M   d  0 (18) NA v  Pmax Herein M is representing the magnification of the chosen objective and NA is the corresponding numerical aperture. The ratio of these values should yield a larger or at least equal value compared to the ratio between the pinhole diameter d0 times the mathematical constant π and the detector radius vPmax, as well as the excitation wavelength of the laser λ. vPmax can vary from below 0.5 to values up to 4. It has to be taken into account that the highest lateral resolution can be achieved with values below

0.5. Usually vPmax is around 2.5 in order not to have a loss z direction. Values of 4 or even higher just leave the resolution of a conventional microscope. [1, 24, 25]

3 Materials and Methods

3.1 Materials

3.1.1 Spectral reference samples For a full identification and characterization of the Raman measured samples, it is necessary to generate a Raman spectral database of important compounds. A discussion regarding to the typical vibrations of the various compounds can be found in 3.2.4. In this chapter just the origin and the preparation of the different reference samples will be discussed. Calcite is one of the most important materials found in the exocuticle of isopods, but also as a major component of the sea urchin tooth. Therefore, reference samples in order

34 to determine the characteristic Raman peaks, but also to reveal the orientational properties of the calcite crystals via Raman spectroscopy, are needed. Small geological calcite crystallites were used for calcite crystal orientation and identification. The crystals were broken out of a large calcite mono-crystal, generating smaller calcite units. The calcite sample was measured with different incident laser light orientation. In order to get the most intense Raman signal, the crystal was mounted along its c-axis. Amorphous calcium carbonate (ACC) is an important material in the formation of calcite, but also has a supportive function in the endocuticle of the isopod tergite cuticle. As an ACC reference and for structural investigations, sternal ACC deposits of Porcellio scaber were investigated, which were provided by Andreas Ziegler of the university in Ulm, Germany. The samples were prepared as described in 3.1.2. Phosphates can be found within the endocuticle of some isopod species as well. Therefore, reference Raman spectra of phosphates are needed for their identification. As it is not totally clear, which kind of phosphates are used in the isopod tergite cuticle, several different kinds of phosphates were characterized. Synthetic amorphous calcium phosphate (ACP) and hydroxyapatite (HA) references were purchased from Sigma Aldrich. Biogenic ACP was measured using pleoventral phosphate deposits of Tylos europaeus, again provided by Mr. Ziegler. The ACP deposits were prepared according to 3.1.2. Geological samples of dolomite and magnesite were measured to observe the peak shift of calcium carbonate due to an increasing Mg2+ incorporation. Furthermore, aragonite, a crystalline calcium carbonate polymorph was measured. The samples were gratefully provided by Mrs. Erika Griesshaber of the LMU in Munich, Germany. The received samples were polished and prepared ready for use. As chitin is one of the most important components, chitin purified out of crab shell chitin was used as reference sample. Furthermore, as organic reference samples, the proteins bovine serum albumin (BSA) and proteins (L-tyrosine, L-histidine, L-aspartic acid, L-glutamic acid) as well as phospho-proteins (O-phospho-L-tyrosine, O-phospho- L-threonine) purchased from Sigma Aldrich were analyzed. For all of the aforementioned components, a uniform integration time of 1 s, accumulating 10 spectra for each measurement was chosen. The laser power was adjusted to 20 mW.

35

3.1.2 Isopod tergite-, joint head- and eye-cuticle Samples were provided by Bastian Seidl, Sukhum Ruangchai and Andreas Ziegler of the University in Ulm, Germany. The samples were pretreated as described in literature [13]. Isopods were dispatched with a 12.5% glutaraldehyde solution. Afterwards the interesting part of the isopod was dissected and prefixed. Specimens were mounted in tangential or sagittal fashion on aluminum or acrylic glass holders using a cyan acrylic gel. The samples were polished by Ultramicrotomy (Leica Ultracut) at ambient temperature, starting with a freshly prepared glass knife at cutting steps of 1 µm at an angle of 45°. Cutting speed is kept quite low at 1 mm/s as samples might break of the holder if the cutting speed is too high. Further steps in order to remove scratches from previous cutting procedures were made at 500, 250 and 100 µm step sizes. For fine polishing the glass knife is replaced by a diamond knife (Diatome) starting at 100 µm cutting steps with 20 iterations. Then the diamond knife is cleaned with isopropanol and the cutting step size is reduced to 50 µm with the same procedure followed by 20 µm step size. Afterwards the sample is stabilized by rinsing it with 100 % methanol for one minute to avoid CaCO3 crystallization. Methanol herein replaces traces of rest water on the sample and therefore avoids further reactions on the sample surface. Samples are dried at room temperature. Typical parameters for Raman spectral mapping were in the range from 0.5 s to 1 s per spectrum in the case of the tergite cuticle samples. Laser power was chosen between 10 and 15 mW at a step width of 400 nm between each spectrum. In the case of the joint head cuticle, typical parameters are 0.5 s integration time per spectrum at 5 mW laser intensity. The same intensity was chosen for the high sensitive cornea cuticle of the eyes of Ligia oceanica as well as Sphaeroma serratum using an integration time of 1 s.

3.1.3 The sea urchin tooth of Paracentrotus lividus Specimen of the sea urchin species Paracentrotus lividus were collected in the Mediterranean Sea at the island Elba. The animals were kept alive until preparation in the laboratory. The masticator apparatus was removed and the teeth were glued and prepared as mentioned in section 3.1.2. Typical parameters for Raman mapping of the sea urchin tooth are 1 s integration time at 20 mW laser intensity using a step width of 400 nm between each spectrum.

36

3.2 Methods

3.2.1 Confocal Raman microscopy Confocal Raman microscopy measurements were carried out on a WiTec Alpha 300AR+ system as illustrated in Figure 6. The system is a combination of a confocal Raman microscope and atomic force microscope. The Raman system is equipped with an Nd-Yag laser (1) operating at a wavelength of 532 nm. The laser intensity can be varied from 0 to 30 mW dynamically. The laser is coupled with the microscope unit via a single mode fiber (2). The incoming light passes a polarizer filter, where the polarization direction of the laser beam can be changed in x and y direction between 0° and 180° and enters the confocal switch between the laser and a white light source (6) for optical microscopy. The objective turret (7) can be equipped with several microscope objectives (8) as mentioned in 2.5.1, which focus the light onto the sample. As the microscope works in backscattered geometry the light again passes the objective and gets focused on the pinhole after passing another objective lens. The functionality of a confocal microscope was already described in 2.5.5. A holographic edge (9) filter ensures a reduction of the Rayleigh peak having a cutoff at 90 cm-1. As almost all Raman systems the WiTec Alpha 300 AR+ is also measuring Stokes scattering. For the investigation of the polarized laser beam, an analyzation unit is equipped (10), which just allows the light having a certain polarization direction (0-180°) to pass. The pinhole size is determined via the diameter of the mounted multi-mode fiber (11), which guides the laser beam to the lens based spectroscopy system (12) having a transmission efficiency of 60%. Fiber diameters of 100, 50 and 25 µm are available. Within the spectroscopy system, the light first passes a collimating lens (13) before it interacts with one of the two equipped gratings (14) with 600 and 1800 grooves/mm. The gratings therefore ensure different spectral resolution of 3 and 0.9 cm-1, as well as a spectral range of 3700 and 1150 cm-1. The spectral center can be changed dynamically offering a wide spectral range. After passing a focusing lens (15) the light gets projected on the back illuminated CCD camera (DV401-BV) which operates at a temperature of -60°C (16) using a Peltier element. The camera is connected to a PC (17) for data acquisition. Furthermore a second slider (18) is equipped within the microscope unit guiding the white light to the optical camera (19), if optical mode is applied. The focusing process is

37

performed via a z-stage (20), which operates with a step motor having a minimum step size of 10 nm up to a maximum of 30 mm. For sample positioning, there are two different systems available. First, for coarse xy alignment and large area measurements, there is the sample positioning unit (21), which has a travelling range of 20 mm in both directions. Second, for exact positioning as well as very small mapping scans, there is a piezo-driven scan stage (22) having a travelling range of 300 µm in both directions, which is mounted on top of the sample positioning unit. The piezo driven stage allows loads with a maximum weight of 500 g. The whole microscope part is mounted on top of an active vibration isolation table (23) in order to eliminate external vibrations. The room temperature is kept at 25°C constantly, as molecule vibrations are sensitive to temperature as mentioned in section 2.4.1. The system is operated with the WiTec Control software and for data evaluation there is the WiTec Project Plus software offering different data evaluation methods, which will be described in the next section.

12 11 13

14 16 15 17 10

2 3 9 1 4

6 5 7

8 20 22 21 23 Figure 6: Scheme and image of the WiTec Alpha 300 AR+ system showing the most important parts of the system. The scan stages open a wide field of application and measurement possibilities. First of all there is of course the possibility to do single point measurements. Second, line scans can be performed either in xy or z direction as illustrated in Figure 7A and B. Furthermore, a line scan in all 3 dimensions is possible. A further possibility is of course mapping of small and larger areas either in xy plane as shown in section C or xz (section D) as well as yz as shown in section E of Figure 7. The mapping methods

38 described in section C and D are also known as depth scans. Furthermore there is the possibility to do a certain number of xy maps at different z coordinates generating a quasi 3D map of the sample, which is called stacked scan (F). If the surface of the sample is not smooth, but is tilted (Figure 7G), there is the possibility to do mapping with a certain tilt. This tilt is calculated by the instrument after teaching it certain points of the area of interest. If the topography of the sample becomes even more difficult, there is the possibility to perform a mapping scan by application of 5 times 5 surface point corrections. The predetermined area herein is split in 5 lines, each of which having 5 points. In each point, the focus has to be adjusted manually. Afterwards the system calculates the topography and performs the map scan. [1]

z z A) B)

x x

z z y y C) D)

x x

z y z y E) F)

x x z y y z G) H)

x x y y

Figure 7: Measurement possibilities offered by the WiTec 300 AR+: A) line scan in xy direction; B) line scan in z direction; C) mapping in xy direction; D mapping in xz direction; E) mapping in yz direction; F) stacked scan (multiple xy map at different z position); G) mapping of a tilted sample via tilt correction method; H) mapping of difficult topography samples via 5x5 surface point correction.

39

3.2.2 Instrument calibration The calibration is typically done using silicon wafer, because silicon has a very characteristic peak located at 521 cm-1 originating from the Si-Si vibration. Silicon is a standard calibration material. Furthermore it has to be ensured that the center of the Rayleigh peak is at 0 cm-1. If this is not the case, a calibration of the grating might be necessary. [30, 31]

3.2.3 Data evaluation After performing one of the Raman experiments as described in 3.2.1 further processing of the data is necessary before a reasonable interpretation can be started. As already mentioned, phenomena like fluorescence or cosmic rays might disturb the measurement making data preprocessing like baseline correction or cosmic ray removal essential. After the preprocessing several methods are applied for data interpretation such as peak integration, Gauss fits in order to determine peak positions, and FWHM (full width at half maximum of the peak). More complicated multivariate analysis like Cluster analysis are used for Raman maps in order to distinguish several areas having similar, but not equal composition.

3.2.3.1 Cosmic ray removal High energy particles from space are interacting with atoms and molecules within the atmosphere yielding highly charged particles, which are also called mesons. These mesons are decaying into muons, which are named cosmic rays if they are able to reach the surface on earth. These rays are able to interact with a CCD camera and generate a signal, not originating from the measured sample itself. The peaks coming from cosmic rays are having a very low broadness but very high peak intensity. The FWHM of sample peaks is usually higher than 3 cm-1 compared to peaks from cosmic rays. In order to remove cosmic rays, the base of the peaks is investigated as common Raman peaks are having a broader base similar to Lorentzian curves. [1]

3.2.3.2 Baseline correction Although confocal Raman microscopy offers a high precision of the measured sample spot, some background effects like fluorescence may yield an increased baseline, which makes the interpretation more difficult. Therefore, a correction of the baseline is

40 necessary. The most useful method is the application of a polynomial of order 5 or even higher. The interesting peaks have to be excluded in the calculation of the polynomial curve via masking. The rest of the data set is used to determine the polynomial curve. Everything below the polynomial is considered as background and subtracted from the original spectrum afterwards. [1]

3.2.3.3 Raman spectral image generation / Univariate data analysis As described in 3.2.1 Raman spectral mapping can be performed in different spatial directions. For the spectral mapping itself, a certain area of interest has to be chosen. The next step is the definition of an array of points within this area via points per line and lines per image as illustrated in Figure 8A defining the spatial resolution of the map. In each of these points, a full range Raman spectrum is acquired. But the limiting lateral and axial resolution as discussed in 2.5.1 and 2.5.2 should be kept in mind to avoid oversampling, which means that the step size from spectrum to the next is way smaller (5-10 times) than the optical achievable resolution. [1] Another aspect regarding the spatial resolution are the number of spectra and the resulting acquisition time, which increases quite fast as following examples illustrate. As an example a 50x50 µm mapping scan shall be mentioned. The integration time for each spectrum shall be 1 second. Moving from 1 µm step size to 400 nm, the duration of the measurement increases by a factor of 6.2. The increase in measurement time becomes even more drastic moving from 400 nm step size to 40 nm in case of oversampling. This leads to an increase of measurement time of a factor of 10000 if the same setting is applied.

Table 3: The connection between spatial resolution, the number of spectra and the resulting time in case of a 50x50 µm Raman spectral map at an integration time of 1 second.

required time for step width number of spectra mapping

1 µm 2500 ~42 min

400 nm 15625 ~4h 25 min

40 nm 1562500 ~434 h 2 min

41

After the acquisition of the spectral map cosmic ray removal and background correction are necessary before a further decent treatment of the data is possible. Generally, two classes of data evaluation methods are used. Univariate methods like the sum or integration of a certain peak are dealing with just one property of each spectrum of a Raman map. For sum integration as an example, an interesting peak has to be chosen (Figure 8B) and the calculated value of this peak is plotted at its corresponding position using an intensity color code. That means, the higher the brightness of the data point, the higher the intensity of the peak at this point, which is depicted in Figure 8C. A certain drawback of peak integration is the fact that the routine is not able to distinguish between several components, yielding an integral over several peaks in some cases. Therefore, it is useful to investigate different peaks of a certain component, generating more than one false color image out of the Raman spectral map in order to avoid these problems. Furthermore, orientational properties in crystalline materials are an issue, as the signal of the crystalline material changes with the direction of the polarized laser beam regarding to the lattice orientation of the material. [1]

A) B) C)

Figure 8: Raman spectral mapping: A) the application of a point array on the area; B) Application of an integral on an interesting peak in each spectrum of the map; C) The resulting intensity color coded Raman false color map.

The peak position is a crucial information on compositional changes within crystalline materials as an example. In terms of biocomposite systems, this can be utilized as there 2+ are often systems containing calcite (pure CaCO3), in which Mg ions due to structural modifications are incorporated into the Bravais lattice of calcite. The librational lattice vibration of calcite for instance gets shifted from 282 cm-1 to 302 cm-1 for dolomite -1 (Ca,Mg(CO3)2) and 331 cm for magnesite (MgCO3). Therefore, a shift of the librational vibration of calcite to higher wavenumbers comes along with an increased incorporation of Mg2+ ions within the calcite crystal lattice and the determination of the

42 peak position via peak fitting is necessary to estimate the amount of Mg2+ incorporation. [12, 32] Despite of simple peak integration, further univariate analysis methods are quite useful like the determination of peak widths. The full width at half peak maximum (FWHM) of a certain peak is a good method for the discrimination of different materials, but also in the discrimination between crystalline and amorphous materials, as amorphous materials are having a quite higher FWHM compared to the crystalline analogue. [1] In the case of calcium carbonate for instance, the FWHM for the carbonate stretching vibration increases from 20 cm-1 for the crystalline form (calcite) up to 32 cm-1 or even higher for ACC. Fitting filters, a different kind of univariate analysis methods, offer a wide field of applications as it is possible to determine peak positions, FWHM and peak intensity via one fitting procedure. Common fitting curves are polynomials, exponential curves, Gaussian-, Lorentzian-, Voigt- and Pseudo Voigt curves. The selection of the fitting procedure strongly depends on the shape of the investigated peak and sometimes different types of fitting curves have to be applied in order to get the best result. [1]

3.2.3.4 Multivariate data analysis In contrast to univariate analysis methods, multivariate methods are using the whole spectrum or multiple selected peak regions for evaluation. Similar to univariate data analysis methods, also intensity color coded generated Raman maps can be generated. The two most prominent methods are principal component analysis (PCA) and cluster analysis. PCA is a good method in data reduction as it is able to reduce the data set to a few dimensions, which in many cases correspond to the number of compounds within the measured sample. Other spectra are simply linear combinations of these compounds. For more detailed information, a closer look into literature is advised. [1, 33] In the case of this work, cluster analysis is the method of choice, because it not only allows the discrimination of different compounds, but also mixtures of several compounds and it is also useful in the differentiation of a single compound having different orientation as not only the spectra, but also the intensities of the peaks are compared within this method. A cluster analysis is a kind of a sorting method, which compares all spectra searching for similarities between the whole or selected regions of

43 a spectrum within the whole Raman spectral map. Areas with similarities are outlined as a component and an average spectrum is calculated for each cluster, which can be clustered further if it is necessary due to orientational or compositional reasons. [1] Each recorded spectrum consists out of 1024 pixel, which is predefined due to the number of pixels within the CCD camera. Each pixel is having its own axis as it can have different intensities due to the Raman measurement and therefore each spectrum consists out of 1024 dimensions. Additionally, in terms of a Raman map, which shall be 50x50 µm in example. Applying a spatial resolution of 1 µm yields 2500 spectra, which are located within these 1024 dimensions. The cluster analysis tries dependent on the chosen method to group these points according to the distance between the various points. The distance between spectra having less in common will be higher than for similar spectra and therefore will be identified as different components yielding different clusters. [1] But before a decent cluster analysis can be performed, some pretreatment of the data is necessary. Again, the first step is to perform a background subtraction in order to remove data due to fluorescence, which could influence the cluster analysis. Another pretreatment is a data reduction, where n pixels of each spectrum are averaged yielding a much more compact spectral data set. As a pre transformation, either a simple derivation of the data or a Savitzky Golay derivated (Savitzky Golay smoothed derivation) are possible. The method of choice for the clustering process itself is K- means clustering, either using the Manhattan or Euclidean distance mode. In terms of K-means clustering the number of clusters has to be determined prior to the clustering process, yielding a cluster tree, which can be split into further sub clusters. [1] The distance between two points according to the Euclidean method is illustrated in equation (19). Herein k and l are the two investigated points, j is the number of measurements, which means that in the case of xkj, we are looking at the jth measurement of sample k. The resulting values of the calculated distances are compared and the smaller the values, the more equal are the data points and therefore the spectra. The Euclidean distance calculation can also be solved in a matrix, which results in an Euclidean distance matrix. More details can be found in literature. [33]

44

J 2 dkl   (xkj  xlj ) (19) j 1

The Manhattan distance differs in its algorithm (equation 20) and therefore also the calculated distances, which will always have greater values compared to the Euclidean algorithm. [33]

J dkl   xkj  xlj (20) j 1

A graphical overview of these two different modes can be found Figure 9A in terms of the Euclidean distance determination and 9B for Manhattan distance determination. As mentioned previously, the distances, if the Manhattan algorithm is applied are higher compared to the Euclidean due to the point distance evaluation algorithm. [33]

A) B) k k

l l

Figure 9: The different ways in point distance calculation: A) Euclidean distance determination; B) Manhattan distance calculation.

Now as the distances between the data points are known, an iterative algorithm is used to find data points with similar point distances. At first random point (number of points should be equal to the estimated components) values are set and the calculated points are assigned to the nearest random point. For a second iteration, the first step is the calculation of new centers, having a better correlation to the first grouped points. Some data points might be moved to a different center as they are now closer to another randomly set point. This procedure is performed 4 times in total and the points assigned to specific random point are presented as a cluster. [1, 33]

45

3.2.4 Reference spectra and interpretation As mentioned previously, the isopod cuticle contains several different components, such as calcite, ACC, chitin and phosphates. Therefore, it is necessary to measure reference spectra in order to make a good peak assignment. The reference spectra are depicted in Figure 10 with an interpretation of the most important vibrations shown in Table 4. All reference samples were measured using 1 s integration time and an accumulation of 10 spectra. The grating with 600 g/mm was chosen to cover the full range up to 3700 cm-1.

Although calcite and ACC are having the same chemical composition (CaCO3), there are still several attributes, which allows discrimination between the two components. At first calcite is having two vibrations at 154 cm-1 and 281 cm-1 as translational and librational external lattice vibrations, whereas in the case of ACC just a broad peak in the range from 60-300 cm-1 can be observed. Generally, it can be stated that all peaks from ACC are having a larger full width at half maximum (FWHM), which is due to the amorphous character of ACC. Despite of a shift of the carbonate stretching vibration from 1088 cm-1 for calcite to 1085 cm-1 for ACC, an increase in the FWHM of the carbonate stretching vibration from 20 to 32 cm-1 for ACC can be detected. As the ACC is from biogenic origin, some other typical vibrations, such as C-H stretching vibrations and O-H stretching vibrations can be identified. [10-12, 34] Despite of the inorganic components, organic components can be localized additionally within the isopod cuticle, which mainly is a chitin protein framework. The most prominent component is chitin and therefore a chitin reference is necessary for a fully identification, which is illustrated in Figure 10C. As chitin is a polymer of 2-acetoamido- 2-deoxy-D-glucopyranose, different chain lengths as well as the degree of acetylation can vary. If chitin is completely deacetylated, the molecule is labelled chitosan instead. Chitin itself can occur in two main different structural units, the helicoidally arranged α- chitin and β–chitin, which is arranged in beta pleated sheets. Sometimes a mixture of both chitin species can occur, which is named γ-chitin then. The most abundant moiety is α-chitin, which can be found in yeast and fungal cell walls, crabs, krills, shrimp shells and isopod cuticle and therefore is of great importance for this work. [35] Within the range of 230-420 cm-1 some amidic (amide VII) as well as out of plane ring modes can -1 be localized. An overtone vibration of –NH2 can be identified at 452 cm followed by

46 further amidic vibrations (amide IV= O=C-N deformation, V and VI) from 482 cm-1 to 670 cm-1, which are again out of plane motions. At 710 cm-1, a very weak vibration originating from CH2 rocking is found. Peaks with higher intensity can be found within the highlighted region in Figure 10C number 10 which are more helpful in the identification of α-chitin. The two peaks at 900 cm-1 and 955 cm-1 are assigned to amide III vibrations. The broad peak at 1057 cm-1 belongs to C-N and C-O stretching vibrations. At 1117 cm-1 C-O stretching vibration resulting from COH groups, as well as C-C stretching vibrations are localized. Peak number 13 in in Figure 10 is representing antisymmetric ring modes. The region highlighted as 14 contains various -1 -1 CH-vibrations, namely CH2-wagging (1330 cm ), CH and CH2 -bending (1380 cm ), as well as another amide III vibration at 1268 cm-1 and an amide II vibration at 1460 cm-1. Amide II and III are mainly originating from N-H bending and C-N stretching vibrations. One of the most characteristic amide vibrations are depicted in section 15, which contains the C-N stretching vibration and N-H bending vibration at 1635 cm-1 and the C=O stretching vibration at 1660 cm-1. Section 16 represents C-H stretching (2895 cm-1) and asymmetric stretching vibration (2935 cm-1). Very characteristic is the broad N-H stretching vibration at 3280 cm-1 (17) as well as the O-H stretching vibration at 3450 cm-1 (18). [36, 37] Further important components in biocomposites are phosphates. Their particular function in terms of isopods is not fully understood up to now but there is a lively scientific discussion about it. One question is why some isopod species are lacking phosphates in their cuticle. Nevertheless also phosphate reference samples are necessary in order to identify the correct phosphate component. Therefore, synthetic hydroxyapatite (HA, Ca5(PO4)3(OH)) and biogenic amorphous calcium phosphate

(ACP, Ca3(PO4)2) originating from pleoventral phosphate deposits of Porcellio scaber were characterized as depicted in Figure 10D,E and Table 4. Hydroxyapatite is exhibiting 5 different vibrations. The most important one is the phosphate stretching vibration at 966 cm-1, which gets shifted to 957 cm-1 in the case of ACP and is also described that way in literature. Apart from the shift of the peak position, peak broadening in the case of ACP (FWHM up to 47 cm-1 instead of 21 cm-1 for HA) can be observed, which is due to the amorphous character of the material. HA additionally

47 exhibits an O-H vibration at 3573 cm-1 and biogenic ACP is having C-H stretching vibrations from 2800-3100 cm-1 proving its biogenic origin. [38, 39] For a reasonable evaluation of Raman spectral maps several peaks are integrated in order to illustrate the distribution or orientation of several components as already outlined in section 3.2.3.3. The most important peaks are highlighted in Figure 10, already showing the color used for intensity color coding of the Raman spectral maps later on. A summary can be found in Table 5. As the integration of the carbonate stretching vibration (1050-1130 cm-1, orange color code) yields the total amount of carbonate, there is no possibility to distinguish between calcite and ACC. Additionally, the red color coded calcite librational vibration (220-320 cm-1) is integrated allowing a discrimination of calcite and ACC. Phosphate is represented in cyan (930-980 cm-1) by integration of the phosphate stretching vibration and organic material via integration of the C-H stretching vibrations in the range of 2800-3100 cm-1, having a green color code.

A) 1 2 3 4a 5 6 calcite

B) ACC 7 4b 8 9

C) 11 1213 14 15 16 α-chitin 17 18 10

D) HA 19 20 21a 22 23

E) ACP 24 25 21b 26 27

Figure 10: The reference spectra used for identification: A) calcite of geological calcite single crystal; B) biogenic ACC out of sternal ACC deposits of Porcellio scaber; C) alpha Chitin out of crab shell; D) synthetic hydroxyapatite (HA); E) ACP out of pleoventral phosphate deposits of Tylos europaeus; The colored peaks are used in the case of Raman spectral mapping to represent a distinct peak or component.

48

Table 4: Peak characterization of the reference spectra as shown in Figure 10. Literature references are listed.

peak peak position component type of vibration (mode) number (cm-1)

1 154 translational vibration

2 281 librational vibration

calcite 3 715 in plane bending [10-12, 34] 4a 1088 symmetric stretching vibration

5 1439 antisymmetric stretching

6 1752 out of plane bending (overtone)

7 90-300 broad characteristic ACC vibration

ACC 4b 1085 carbonate stretching vibration [11, 12] 8 2800-3100 C-H stretching vibration

9 3400-3500 O-H stretching vibration

10 900, 955 amide III vibration

11 1057 br. C-O, C-N stretching vibration

12 1117 C-O str. vib. (of COH group), C-C str. vib.

13 1210 antisymmetric ring modes

chitin 14 1230-1520 CH2 wagging and bending CH-bending [36, 37] 15 1635, 1660 N-H in phase bending and C-N stretching vib., C=O stretching (Amide I)

16 2895, 2935 CH str., CH-asymmetric str. vibration

17 3280 N-H stretching vibration

18 3450 O-H stretching vibration

49

peak peak position component type of vibration (mode) number (cm-1)

19 440 v2 mode

20 590 v4 mode HA 21a 966 symmetric stretching vibration (v1) [38, 39]

22 1050 v3 mode

23 3576 O-H stretching vibration

24 430 v2 mode

25 583 v4 mode ACP 21b 957 symmetric stretching vibration (v1) [38, 39]

26 1080 v3 mode

27 2800-3100 C-H stretching vibration

Table 5: Peak integration ranges and the applied color code used for Raman spectral mapping in order to illustrate the distribution of several components.

integration range represented intensity color type of vibration / cm-1 compound code

calcite librational 220-320 calcite red vibration

carbonate symmetric 1050-1130 total carbonate orange stretching vib.

phosphate 930-980 symmetric stretching phosphate cyan vibration

C-H stretching 2800-3100 organic matter green vibration

50

3.2.5 Orientational properties of calcite The orientation of calcite with its rhombohedral crystal symmetry is important information as the mechanical and optical properties are strongly depending on the orientation of the calcite crystal. Therefore, more knowledge on the orientation of the calcite crystals is very useful. The orientation of crystalline samples for instance can be investigated by means of polarized confocal Raman microscopy. Herein the polarization angle of the incident laser beam is changed with respect to the sample surface and changes in the peak intensities of several peaks can be observed. [34, 40] For calcite, the c-axis is the most important one as it is the axis, where the carbonate ions are aligned and therefore the most intense carbonate stretching vibration can be detected there as shown in Figure 11. In consequence, the calcite crystal was mounted on the stage below the microscope unit of the Raman microscope in a fashion that one of the1014 faces of the calcite rhombohedra were perpendicular compared to the z- direction of the incident laser light and parallel to the polarization of the incident laser light P0° (Figure 11A). Changing the polarization by 90° leads to a strong increase in the peak intensity of the carbonate stretching vibration (app. factor 3) as shown in Figure 11B. On the contrary in case of the calcite librational vibration, just small changes in the peak intensity can be observed due to a polarization change. The peak intensity of the carbonate stretching vibration changes continuously when the polarization is changed stepwise from -90° to 90°. For each spectrum a ratio between the integral intensity of the librational vibration and the carbonate stretching vibration is calculated introducing the carbonate ratio (CR) as a quantity to estimate the calcite crystal orientation (equation (21)). [41]

220 320cm1 CR   (21) 1 1050 1130cm For a more detailed investigation of the orientational dependency of the CR, it is necessary to rotate the polarizer stepwise from -90° to 90°. Steps of 15° were chosen and the results are shown in Figure 12. The integral intensity of the calcite librational vibration increases slightly towards 0° polarization compared to the carbonate stretching, which decreases towards 0° polarization. The CR is calculated using the integrated intensity values of the spectra as shown in equation (21) yielding values

51 starting at 0.4 at 90° polarization up to 1.2 at 0° polarization angle. This implies that the lower the CR value, the more the calcite is oriented in the c-axis of the calcite crystal, which is also in good agreement to EBSD measurements as also mentioned in the literature. [41]

B) A) P0°

P90°

Figure 11: The Bravais lattice of calcite and polarization dependent calcite vibrations: A) Calcite Bravais lattice showing the orientation of carbonate and calcium ions. The c-axis is highlighted and the laser polarization directions with respect to shown crystal plane are depicted. B) Two calcite spectra measured along the c-axis at 0° and 90° laser polarization. The calcite librational vibration at 281 cm-1 and the carbonate stretching vibration at 1088 cm-1 are illustrated graphically.

B) A)

Figure 12: Changes of the integral intensities in calcite due to different laser polarization angles. A) Integral intensity of the calcite librational vibration (black curve) and the carbonate stretching vibration (red curve) depending on the laser polarization angle. B) The CR value for each polarization angle using the values of A) for ratio determination.

52

3.2.6 Mg-calcite and the determination of the MgCO3 content Another phenomenon is the substitution of Ca2+ by Mg2+ within the Bravais lattice of calcite. Magnesium incorporation has a strong effect on the physical properties as well as a change of the Raman spectrum. The FWHM of a peak is a good quantity to characterize a material, as crystallinity, structural defects as well as crystallite size, but also the substitution of elements is causing changes in the absolute values. The FWHM of the carbonate stretching vibration is increasing starting with calcite (6 cm-1) to 8 cm-1 for dolomite and 14 cm-1 for magnesite if the 1800 g/mm grating is applied. A plausible explanation for this issue is the size of the cation, as Ca2+ is larger compared to Mg2+ and therefore the Ca-O bond for calcite (2.36 Å) is way larger but also weaker as the Mg-O bond for magnesite (2.1 Å). Therefore, the vibrational frequencies are increasing with an increasing Mg2+ substitution, which means that the carbonate related peaks are shifted to higher wavenumbers within the Raman spectrum. In the case of geological calcite the carbonate stretching vibration gets shifted from 1087 cm-1 to 1098 cm-1 for dolomite but is again lower for pure magnesite (1096 cm-1). The corresponding spectra are depicted in Figure 13 with a fully interpretation shown in Table 7. The shift to lower wavenumbers from dolomite to magnesite mainly has to do with a decrease of the disorder in the Bravais lattice as only one type of cations are present forming a more symmetrical Bravais lattice. Although there are slight differences in peak position and FWHM between geological and biogenic material, it is nevertheless possible to estimate the content of MgCO3 within the Mg-calcite region. A comparison with literature allows the conclusion that it is valid to estimate the MgCO3 content via determination of the peak shift in comparison to pure calcite and dolomite. A linear correlation concerning the peak shift of the carbonate stretching vibration and the increasing Mg2+ content in calcite was found in literature. Therefore it is possible to determine the MgCO3 content of any unknown Mg-calcite sample just by determination of the center peak position of the carbonate stretching vibration peak. [12, 14, 42] It is of course inevitable to determine the peak position of references, in order to know the boundary conditions of the used Raman system as the wavelength of the applied Raman laser is having an influence on the peak positions. If these things are kept in mind, results, which are in good agreement with already published XRD measurements,

53 can be achieved as shown in Table 6 for isopod tergite cuticle samples. The regions used for average spectra determination were solely taken out the exocuticle of the various isopods. Gauss fits were used for peak position determination.

Table 6: Determination of the MgCO3 content in Mg-calcite due to the peak position of the carbonate stretching vibration of different isopod species (exocuticle). A comparison with XRD results is shown.

Raman shift carb. MgCO mole% MgCO mole% sample origin 3 3 Str. vib. / cm-1 by Raman by XRD [14]

geological calcite 1088.1 0 0

Tylos europaeus 1089.0 4.17 4.29

Helleria brevicornis 1088.3 0.96 1.02

Porcellio scaber 1088.9 3.70 3.63

Armadillidium vulgare 1088.8 3.24 3.30

Sphaeroma serratum 1089.3 5.56 5.64

geological dolomite 1098.9 50 50

3.2.7 Polymorphs of calcium carbonate Aside from the determination of Mg2+ within calcite, Raman spectroscopy is a good tool to discriminate also polymorphs of certain materials. In the case of calcium carbonate, there are three different crystalline polymorphs. Calcite, aragonite and vaterite can be distinguished due to their characteristic Raman vibrations. Calcite has been shown already, but as a second example aragonite shall be mentioned. Aragonite is having orthorhombic space group, which leads to more translational and rotational lattice modes in the region between 100-350 cm-1 ((13) in Figure 13D and Table 7). -1 -1 Characteristic for aragonite is the doublet of the v4 vibration at 704 cm and 717 cm (14). A vibration just allowed for aragonite due to its crystal geometry is located at -1 853 cm (v2 band, (15)), showing very low peak intensity but nevertheless can be used as further criterion to distinguish between aragonite and calcite. The peak center for the carbonate stretching vibration of aragonite is located at 1086 cm-1 (16). [43]

54

A) calcite 4 2 1 3

B) dolomite 8 6 5 7

C) magnesite 10 12

9 11

D) aragonite 13 16

14 15

Figure 13: The Raman spectra of different carbonate species within a range from 0 to 1150 cm-1. It has to be noted that all spectra were recorded with the 1800 groves/mm grating. All samples are of geological origin: A) calcite (CaCO3, rhombohedral); B) dolomite (Ca,Mg)(CO3)2; C) magnesite (MgCO3) and D) aragonite (CaCO3, orthorhombic). The type of vibrations can be looked up in Table 7.

Table 7: Fully interpretation of the Raman spectra as shown in Figure 13. References used for the interpretation are shown for each component.

peak position component peak number type of vibration (mode) / cm-1

1 154 translational vibration

calcite 2 281 librational vibration [10-12, 34] 3 715 in plane bending

4 1087 symmetric stretching vibration

55

peak position component peak number type of vibration (mode) / cm-1

5 175 translational vibration

dolomite 6 299 librational vibration [12] 7 725 in plane bending

8 1098 symmetric stretching vibration

9 212 translational vibration

magnesite 10 329 librational vibration [12] 11 738 in plane bending

12 1096 symmetric stretching vibration

13 153, 181, 206, lattice modes of aragonite 249, 262

aragonite 14 704, 717 doublet of in plane bending (v4) [43] 15 v band (only allowed for 853 2 aragonite)

16 1086 symmetric stretching vibration

56

4 Investigation of different isopod cuticle systems 4.1 General aspects on isopods and their cuticle

Arthropods are belonging to the group of invertebrates, which have jointed appendages, a segmented body and a hard mineralized outer exoskeleton. Arthropods can be divided into crustaceans, insects and arachnids. Isopods belong to the group of crustaceans, which differ in quite wide ranges in their size. There are species, which are about 300 µm in size on the one hand, like the group of microcerberidae, which live in the sea but also on sandy beaches and on the other hand there are isopods having a size of 50 cm belonging to the group of giant isopods (exemplarily Bathynomus giganteus). Depending on the habitat and the defense mechanism of the species, thickness and composition of their outer tergite cuticle are varying. The tergite cuticle of isopods is of particular interest in terms of biomimetic as it combines quite diverse mechanical properties like a hard outer layer against predation, but also a softer inner tissue where muscles can attach. It even works as a barrier against water loss and pore canals for material transport can be identified. The tergite cuticle is arranged strong hierarchically, each level having an important function for the whole cuticle. [13, 15, 16] Isopods have quite diverse habitats, ranging from marine to different terrestrial habitats like sandy beaches, forestral regions and also stony regions. Each species adapts its cuticle for its needs in order to have protection against predation. An overview of the most interesting species is depicted in Table 8. [27, 28, 30] Herein the differences in the habitat of the investigated isopod species are illustrated. Furthermore three different types of defense mechanisms are common, which have an effect on the thickness of the exocuticle. Generally a higher thickness of the exocuticle comes alongside with a higher overall mass. The group of runners therefore is having a thin exocuticle compared to the other groups as they are running away in the case of danger but have way longer legs. The group of rollers is having a thicker exocuticle as they are rolling into a sphere in the case of predation and therefore a thicker exocuticle means higher safety. The third group is the group of clingers, which can attach very close to the surface due to their flat body and cannot be removed from there, but they are also quick in running away. [27, 28, 30]

57

Table 8: Investigated species with their characteristically habitat, the type of defense mechanism and the thickness of the exocuticle.

habitat defense mechanism exocuticle thickness

mainly forestral very thick Helleria brevicornis rolls into a sphere regions (15-20 µm)

very thick Tylos europaeus beach, sandy sites rolls into a sphere (15-25 µm)

chalky or limestone thick Armadillidium vulgare rolls into a sphere sites (10-15 µm)

Stony and forestral Thin and flexible Porcellio scaber runner, clinger regions (app. 5 µm)

marine, rock very thick Sphaeroma serratum rolls into a sphere crevices (up to 30 µm)

4.2 The tergite cuticle of isopods

The overall tergite cuticle is consisting of 7 thoracomeres, as depicted in Figure 14A. Herein the thoracomere 2-5 are of particular interest, as they are consisting of a smooth (anterior part) and a part where antenna, sensilla and microtubercles are attached (posterior part) (Figure 14B). The smooth anterior part herein is important as it is used when the isopod is forming a sphere in case of predation. Usually the anterior part is located beneath the preceding thoracomere when the animal is not forming a sphere for safety reasons. Performing a sagittal cut through a thoracomere reveals the smooth character of the anterior part as well as the surface elements within the posterior part of a thoracomere (Figure 14C). In section D, the major elements forming the classical hierarchical structure of the isopod tergite cuticle are visualized. Although thickness and chemical composition are varying, these four elements can be identified within each isopod tergite cuticle. The most distal section is the epicuticle, which is a layer consisting of waxes protecting the following calcium carbonate crystalline section of calcite named exocuticle. The endocuticle is consisting of a chitin protein fiber network, which is supported by amorphous calcium carbonate as well as other proteins and in the case of some isopod species also phosphate can be localized. The membranous layer is

58 built upon densely stapled chitin protein fibers. Pore canals can be found within the whole framework, which are necessary for the transport of proteins, water and material for calcification. [17, 44, 45]

A) C)

D) epicuticle 1 2 3 exocuticle 4 5 6 pleon 7 endocuticle

membraneous layer B) 30 µm

Figure 14: The tergite cuticle of an isopod showing the pleon and 7 thoracomeres in section A), the surface of a thoracomere showing the anterior and posterior part of a thoracomere (B) and a sagittal cut through a thoracomere in section C, D) the hierarchical arrangement within a tergite cuticle showing several hierarchical levels from the distal to proximal side.

The chitin protein matrix is arranged at least at 7 hierarchical levels as shown in literature [14, 15]. A scheme showing the 7 different hierarchical levels of the chitin protein matrix is depicted in Figure 15. The first step is the generation of N-acetyl- glucosamine chains (Figure 15A), followed by the formation of anti-parallel α-chitin chains (B). The third level are nano-fibrils (C), which are formed out of 18-25 chitin chains in combination with proteins, having a diameter of approximately 3 nm for each nano-fibril. These nano-fibrils cluster to form chitin-protein-fibers of up to 250 nm in thickness as shown in Figure 15D. The long axis of the fibers is arranged the same way for all of the fibers forming horizontal planes. The honeycomb like structure shown in section E originates due to the arrangement of the fibers around the cavities of the pore canals. Theses chitin protein planes are lying upon each other with a slight disorientation originating from the orientation of the chitin fibers forming the well- known twisted plywood like structure, which looks like a helix (section F). The height required to make a rotation of 180° for the chitin protein planes is named stacking

59 height. A microscope image illustrates the stacking of the chitin protein plane for the endocuticle of Helleria brevicornis, which is depicted in section G. [18-20, 44, 46]

C D

A

~3nm B ~10Å 50 nm 200 nm G

F

10 µm

E

Figure 15: The 7 hierarchical levels forming the chitin protein matrix; A) N-acetyl-glucosamine molecules; B) α-chitin chains; C) chitin-protein nano-fibrils; D) chitin-protein fiber bundles; E) chitin- protein planes; F) twisted plywood like structure; G) optical image showing the twisted plywood like structure within the endocuticle of Helleria brevicornis. [20]

As the tergite cuticle of isopods is a combination of several quite diverse mechanical properties like elasticity but also hardness in one work piece, structure and function play an important role. Several components can be identified in the tergite cuticle, but the combination and structure of these materials is the key in forming the aforementioned unique properties. A general overview of the most abundant components can be considered as a good starting point for the spatial localization of these components by means of confocal Raman microscopy. The investigation of the total chemical composition of the 5 species was performed by several methods. The values were provided by ([14, 15]), but the methods shall be described briefly. The mineral content was determined by X-ray powder diffraction to identify calcite. By addition of known amounts of SiO2 to the powdered samples, Rietvield methods allow the determination of the total amount of crystalline calcite within the sample. In order to determine the Mg2+ content in Mg-calcite, the peak positions of the diffraction peaks as well as peak intensity changes compared to pure calcite were investigated. Thermogravimetric

60 analysis methods were used as well as IR and DSC. More details on this work can be found in literature ([14, 15]).

4.2.1 The overall chemical composition of the tergite cuticle for several interesting isopod species As shown in Table 9, the total chemical composition of the 5 investigated species differs in quite wide ranges. Although the most abundant component is ACC for each species, its total amount varies from 35 % for Porcellio scaber up to 54 % for Armadillidium vulgare. Mg-calcite on the contrary only varies from 10.8 % for Armadillidium vulgare to 21.1 % for Tylos europaeus, which also manifests in the thickness of the exocuticle but will be shown later on. Another component is hydroxyapatite. Some species like the marine isopod Sphaeroma serratum are completely lacking phosphates, whereas Porcellio scaber is having a phosphate content of up to 11.2%. Phosphate is indicated as HAP through XRD because the elemental analysis includes a heating step up to 1000 °C and therefore the phosphate originally either consisted of amorphous calcium phosphate (ACP) or phosphorylated proteins of the chitin matrix. This ambiguity has not been cleared up to know and is still part of scientific discussion. The organic matrix is the third most abundant component within the tergite cuticle of the investigated isopod species and is followed by water. [14, 15]

Table 9: The total chemical composition of the tergite cuticle of the 5 investigated isopod species. The values were provided by [14, 15].

Helleria Tylos Armadillidium Porcellio Sphaeroma

brevicornis europaeus vulgare scaber serratum

Mg-calcite 16 % 21.1 % 10.8 % 14.5 % 12.3 %

ACC 52 % 42.3 % 54 % 35 % 52 %

HAP/ACP 3.4 % 5.2 % 9.7 % 11.2 % 0 %

organic 12.2 % 19.3 % 11.7 % 24.8 % 10.5 % matrix

water 12.3 % 11.4 % 9.7 % 8 % 12.9 %

unknown 4.1% 0.7 % 4.1 % 6.5 % 12.3%

61

4.3 Results and discussion

4.3.1 Determination of the local chemical distribution with confocal Raman microscopy As mentioned in 4.2 the tergite cuticle of isopods consists of a hierarchical arrangement of 4 major structural elements, namely epicuticle, exocuticle, endocuticle and membranous layer. A more detailed description on the arrangement of these elements was already given in terms of Figure 14 in 4.2. Herein, a distribution of several chemical compounds like calcite, ACC, phosphates and chitin protein fibers can be observed. As a first example, normalized average spectra of these 4 elements were calculated (Figure 16) for the tergite cuticle of Helleria brevicornis. The uppermost layer (epicuticle) is consisting out of waxes, which manifests in notable C-H stretching vibrations as well as amidic vibrations in the region around 1600 cm-1. The epicuticle is a very small layer around 1 µm in thickness and therefore it is no surprise that the following layer (exocuticle) also makes a contribution to the average spectrum of this very small layer. As the exocuticle is composed of calcite, the typical vibrations can be found in the epicuticle as well. Typical calcite vibrations like the carbonate stretching vibration as well as calcite lattice vibrations can be detected. A detailed list of the 6 characteristic calcite vibrations can be found in Table 4 (section 3.2.4). In the endocuticle, three major components can be identified due to the average spectrum in Figure 16. First, there is ACC, which can be discriminated from calcite due to the broad peak feature in the range of 90-300 cm-1. Additionally, no typical calcite lattice vibrations can be found. A further criterion is the peak shift of the carbonate stretching vibration from 1088 cm-1 to 1085 cm-1 in combination with a strong increase of the FWHM from 20 cm-1 to 32 cm-1 as shown in 3.2.4. Furthermore, strong Raman vibrations originating from chitin can be identified. There are characteristic vibrations in the C-H (2800-3100 cm-1), N-H (3280 cm-1) and O-H (3450 cm-1) stretching vibration range, as well as amidic vibrations (1630-1680 cm-1) and C-C vibrations, which are in good agreement to the measured chitin reference sample in 3.2.4, indicating the presence of helicoidally alpha chitin chains (4.2). Aside from ACC and chitin, the band at 957 cm-1 is a strong indication that phosphate is present within the endocuticle. A comparison with the reference spectra in 3.2.4 is a good prove for that.

62

Additional support is found in literature ([14, 15]), as different investigation methods like EDX were used for phosphate determination within the tergite cuticle of isopods as well. The innermost section of each tergite cuticle is the membranous layer (Figure 16). The average spectrum of this region just shows signs of organic material and mainly consists out of chitin protein fibers, as a comparison of the spectrum with chitin from the reference spectra (3.2.4) reveals.

epicuticle

exocuticle

endocuticle

membranous layer

Figure 16: Normalized spectra of the 4 important sections within the isopod tergite cuticle. Helleria brevicornis was chosen as an example: The hierarchical levels starting from the most distal regions are epicuticle, exocuticle, endocuticle and membranous layer.

Now as the composition of each of the sections of the tergite cuticle is known principally, the spatial distribution as well as compositional changes within these regions is of particular interest. Raman spectral mapping over sagittal cross sections allows an investigation of the aforementioned hierarchical levels. The main benefit of the spectral mapping lies in the possibility to discriminate the compounds at high spatial resolution. In order to create a more detailed view on the isopod tergite cuticle

63 composition, 5 different isopod species were investigated with Raman spectral mapping. The reason for this is that also environmental influences on composition and structure are of particular interest, as adaptions in nature are always made due to external influences and vital needs. In terms of Raman mapping, several interesting peaks (according to Table 5) were investigated and corresponding color codes applied to plot the peaks of the certain compounds.

4.3.2 Organic material within the tergite cuticle (epicuticle, chitin as matrix within the endocuticle and membranous layer)

4.3.2.1 The epicuticle as outmost layer within the tergite cuticle of isopods The epicuticle as uppermost layer of the isopod tergite cuticle consists out of waxes and proteins, acting as a barrier against water loss. Crucial for the detection of organic material in Raman spectra is the integration of the C-H vibrations in the spectral region of 2800-3100 cm-1. A green intensity color code is used for the plots of the C-H peak intensity distribution maps (in case of Raman mapping). In Figure 20A, C and E the epicuticle can be detected as a very thin layer between 1 and 2 µm thickness. In the case of Porcellio scaber and Tylos europaeus (Figure 20B,D) the epicuticle is still there but below the detection limit as it is even thinner compared to the other three species. Exemplarily, the average spectrum of the epicuticle of Helleria brevicornis was already discussed in 4.3.1 (Figure 16). Due to the low thickness of the epicuticle it is nearly impossible to get an average spectrum solely of this layer. Most frequently, a combination of epi- and exocuticle as an average spectrum is received.

4.3.2.2 Chitin protein matrix within the endocuticle and membranous layer and its orientational properties The endocuticle of an isopod tergite cuticle is a framework consisting of helicoidally arranged chitin protein fibers as mentioned in Figure 15 (section 4.2), where additionally ACC is incorporated. The distribution of ACC will be discussed in 4.3.3. As polarized Raman is sensitive to changes within the orientation, the helicoidally arrangement of the chitin protein fibers seem to form layers upon each other, which is not the case. This phenomenon can be described by the fiber orientation of the chitin protein fibers. The distance for a twist (turn of 180°) of the chitin protein fiber is named

64 stacking height. [20] Before a determination of the stacking height can be performed, several further investigations regarding to the orientation of the chitin protein fibers are necessary. For a more detailed look on the orientation of the chitin protein fibers, the inorganic components have to be removed in order to exclude external influences onto the Raman signal of the chitin protein fibers. Therefore, exemplarily a tergite cuticle sample of Helleria brevicornis was decalcified. The decalcification procedure was performed using ultrapure water as decalcification agent, which dissolves ACC, but leaves the organic components. The sample was decalcified for one minute using a magnetic stirrer to ensure constant decalcification conditions. Afterwards, the sample was fixed using pure methanol and dried at room temperature. For the investigation of the orientational properties of the chitin protein fibers by means of confocal Raman microscopy, it is necessary to record the spectra with at least two different incident laser light orientations as already described in the materials and methods section. In the case of the decalcified sample 0° and 90° incident laser polarization for the Raman spectral mapping were chosen, which correspond to parallel and perpendicular laser light orientation with respect to the sample surface. As the peak intensity of the C-H stretching vibration shows lower changes to orientational changes of the incident laser light compared to the N-H stretching vibration (3174-3374 cm-1), the latter vibration was chosen. The resulting plots (yellow intensity color code) can be found in Figure 17. At 0° incident laser polarization, it seems that the sample exhibits a layered arrangement because just every second layer shows notable peak intensity. Switching to 90° incident laser light orientation shows the opposite effect. Now it seems that the layers which previously had quite low intensity are having high intensity and vice versa. The only reasonable explanation for this effect is of course the orientation of the helicoidally arranged chitin protein fibers. A change on the orientation of the incident laser light by a certain angle is accompanied by a shift of the peak maxima within the sample, because a different part of the chitin helix will be oriented in the same fashion as the incident laser light and therefore exhibit the peak maximum. This means that at parallel laser light orientation (0° polarization), the parallel oriented chitin fibers will give the most intense signal. A change to perpendicular laser light orientation (90° polarization) therefore yields to peak maxima resulting from perpendicular oriented chitin fibers. Two spectra at 0° and 90° incident laser light polarization were

65 extracted at the positions marked in Figure 17 in order to illustrate the dependency of the peak intensity of the chitin fibers due to the laser light polarization (Figure 18). The position was chosen within the membranous layer as the total chitin spectrum is having a way higher peak intensity compared to the spectra representing the endocuticle. For a further validation, cross sections were calculated along the lines in Figure 17. The calculations were made for both incident laser light orientations, which are shown as a plot (peak intensity versus position) in Figure 19.

0° pol. 90° pol.

8 µm 8 µm

Figure 17: Chitin fiber orientation within the endocuticle and membranous layer of a decalcified tergite cuticle sample of Helleria brevicornis. Two different incident laser light orientations (0° and 90° polarization) were used and the plotted lines were chosen for cross section determination using the corresponding color in Figure 19. The integrated vibration for these two Raman maps is the N-H vibration in the range of 3170-3370 cm-1 using a yellow color code. The cross in each spectrum marks the position of the spectra extracted for comparison in Figure 18.

66

The normalized spectra at 0° and 90° incident laser light orientation show quite a few changes in the chitin spectrum when they are compared to each other. There are changes in the peak intensities of the O-H stretching vibration at 3450 cm-1, which is more intense at 0° polarization contrary to the aforementioned N-H stretching vibration originating from amide in the chitin molecule at 3260 cm-1. The normalized spectra also show a drop in the peak intensity of the C-H stretching (at 2885 cm-1) from 1.0 at 90° polarization to 0.8 at 0° incident laser light polarization. Further changes can be found in the C=O and amide-I region at 1660 cm-1 resp. 1630 cm-1, where a slight increase of the peak intensity at 90° laser polarization can be observed. Just small changes can be found in the range of 1190-1570 cm-1 where CH wagging and bending vibrations occur. The peak at 1110 cm-1 belongs to the C-O, C-C and C-N stretching vibration region and decreases from 0.48 at 0° incident laser light polarization down to 0.29. The amide III´ vibration at 955 cm-1 at 0° pol. is approximately at 0.19 and 0.29 for 90° polarization.

Figure 18: Normalized average spectra as marked via a cross in Figure 17 at 0° (magenta spectrum) and 90° (red spectrum) incident laser light polarization. A full interpretation can be found in Table 4 (3.2.4).

67

Furthermore, small changes at 910 cm-1 can be detected but are not as crucial as the previously mentioned vibrations. Other vibrations below 900 cm-1 do not show a significant change in their peak intensities to be of a particular interest. Looking at the cross sections as illustrated Figure 17, the peak intensity can be plotted as a function of its corresponding position (Figure 19). The distance between two intensity maxima herein corresponds to a twist of 180° by the chitin protein fibers, which correlates to the aforementioned stacking height. A comparison of the cross sections (Figure 19) yields the same result as previously described in terms of Figure 17. The N-H peak intensity maxima at 0° incident laser polarization correspond to peak intensity minima at 90° incident laser polarization and vice versa. Therefore, it is not necessary to measure at two different laser light polarization angles for stacking height determination of the chitin protein fibers.

membranous layer

stacking height determination

endocuticle

Figure 19: Peak intensity distribution of the N-H vibration over the cross sections as marked in Figure 17. Endocuticle and membranous layer are highlighted. The stacking height can be determined via calculation of the distance between two intensity maxima or minima.

68

In the case of Helleria brevicornis the stacking height varies between 2.8 and 3.4 µm, which also applies for the membranous layer although the peak intensities are higher compared to the endocuticle. The collected knowledge on the determination of the stacking height is used for stacking height determination of all 5 investigated isopod species. As just a single incident laser light orientation is necessary, 0° polarization was chosen for all following measurements within this chapter per definition. In Figure 20 the Raman spectral maps are showing the distribution of the organic chitin protein matrix in the tergite cuticle of all 5 investigated isopod species. Herein the C-H stretching vibration was used for map generation as the N-H stretching vibration peak intensity is too weak in non-decalcified samples. Big differences in stacking height and overall endocuticle thickness can be found looking at the 5 different specimens. Average spectra of the endocuticle of all 5 investigated isopod species were calculated and can be found in appendix section 7.1. Aside from the endocuticle also the epicuticle and the membranous layer are shown in Figure 20 as the maps are representing the distribution of all present organic material within the Raman map. Large differences can be found regarding the overall diameter of the cuticle and also the stacking height (determined analogue to Figure 17 and Figure 19) of the chitin protein fibers. All determined parameters of the investigated specimen are summarized in Table 10.

A) B) C) D) E)

8 µm 4 µm 8 µm 8 µm 12 µm

Figure 20: Raman maps showing the distribution of organic matter (integration of the C-H stretching vibration from 2800-3100 cm-1) in the exocuticle for 5 different isopod species using a green intensity color code for visualization: A) Helleria brevicornis; B) Porcellio scaber; C) Armadillidium vulgare; D) Tylos europaeus; E) Sphaeroma serratum.

69

Cross sections were prepared for each specimen in Figure 20 for stacking height determination. The results can be found in Figure 21. In case of Helleria brevicornis the stacking height is in the range between 2.8 and 3.8 µm with an overall endocuticle thickness of 55 µm. Porcellio scaber on the contrary shows quite different behavior, as the C-H stretching vibration peak intensity continuously increases towards the membranous layer, showing no distinct maxima for stacking height determination and therefore could not be assigned. The total diameter of the endocuticle is app. 17 µm. For Armadillidium vulgare, an unsteady stacking height between 1.8 and 2.8 µm for the chitin protein fibers can be detected, having a total diameter of 46.5 µm for the endocuticle. Tylos europaeus and Sphaeroma serratum are quite different concerning the stacking height as it is decreasing continuously towards the membranous layer. In case of Tylos europaeus (overall endocuticle thickness of 47 µm) the stacking height decreases from 5.2 µm down to 2.5 µm. Sphaeroma serratum shows similar changes as the stacking height decreases from 5.9 µm to 2.6 µm towards the membranous layer. The total diameter for the endocuticle is app. 82 µm, which is the largest diameter of all investigated species. Quite remarkable is the transitional region between exo- and endocuticle within the tergite cuticle of Sphaeroma serratum as a higher organic content compared to the other isopod species in the corresponding region can be detected (Figure 20E and Figure 21 for Sphaeroma serratum). The calculated average Raman spectrum shows a high content of chitin present in this transitional region (Figure 22). Interestingly, it seems that a higher chitin concentration can be found there as the typical vibrations for chitin can be identified. But this region is a rather small transitional area between exo- and endocuticle and therefore traces of calcite can be detected within this average spectrum as well.

Table 10: The thickness of different regions within the tergite cuticle of all 5 investigated species. *diameter was determined with underlying samples of Figure 20.

overall diameter of diameter of stacking isopod species thickness of membranous endocuticle* height* tergite cuticle* layer*

Helleria 78 µm 55 µm 2.8-3.8 µm 4.6 µm brevicornis

70

overall diameter of diameter of stacking isopod species thickness of membranous endocuticle* height* tergite cuticle* layer*

Porcellio 24 µm 17 µm not assignable 3.5 µm scaber

Armadillidium 65 46.5 1.8-2.8 6.5 vulgare

Tylos europaeus 67 47 5.2-2.5 6

Sphaeroma 82 58 5.9-2.6 7 serratum

Helleria brevicornis membranous layer endocuticle

epicuticle

Porcellio scaber

Armadillidium vulgare

Tylos europaeus

Sphaeroma serratum

Figure 21: Cross sections over the Raman map of the C-H stretching vibration showing epi- and endo- cuticle as well as membranous layer. The cross sections of all 5 investigated isopod species are shown. The distance between two peak maxima in the endocuticle corresponds to the stacking height of the chitin protein fibers. Increasing distance equals a movement more in the proximal region of the endocuticle. The epicuticle, endocuticle and membranous layer for each species are highlighted using colored boxes.

71

Figure 22: The average Raman spectrum of the transitional region between exocuticle and endocuticle within the tergite cuticle of Sphaeroma serratum, showing a mixed spectrum of calcite and chitin.

The most proximal layer is the membranous layer consisting solely out of organic chitin protein matrix, where no inorganic material can be identified. This is important as the membranous layer is very soft in order to enable the attachment of muscles and to keep the whole tergite cuticle flexible. The most proximal part in the determined cross sections in Figure 21 (red box) can be assigned to the membranous layer. Usually, the membranous layer therefore exhibits higher peak intensities compared to the endocuticle. The reason for this is the missing ACC, which quenches the chitin signal within the endocuticle. The thickness of the membranous layer varies in quite small ranges from 3.5 µm for Porcellio scaber, 4.6 µm for Helleria brevicornis, 6 µm for Tylos europaeus, 6.5 for Armadilldium vulgare to 7 µm in the case of Sphaeroma serratum, which is summarized in Table 10. Average spectra of all 5 investigated species were calculated and visualized in appendix 7.1, as no big differences in the spectra except of the peak intensities could be found.

72

4.3.3 Inorganic components within the tergite cuticle (exocuticle, ACC and phosphate within the endocuticle) Besides the previously discussed organic materials, inorganic materials can be found within the tergite cuticle as well. These materials are playing an important role in the structure and function principle of the whole cuticle composite. Calcium carbonate

(CaCO3) can be found in crystalline (i.e. calcite) and amorphous state named amorphous calcium carbonate (ACC). Additionally, modifications of calcite can be found as different amounts of magnesium are incorporated in calcite altering the physical properties of the calcite layer. Furthermore, phosphates can be detected within the endocuticle but their function is still a matter of discussion in the scientific community and it is still not fully known whether the phosphate is of inorganic or organic origin. [13-21] 4.3.3.1 The exocuticle of the isopod tergite cuticle. The exocuticle solely consists out of calcite or Mg2+ incorporated calcite and represents the hardest part within the whole tergite cuticle framework. As already shown in Figure 14D, the exocuticle is located below the epicuticle but above the endocuticule in a sagitally prepared sample. For the investigation of calcite, the calcite librational vibration is crucial, which is important in the discrimination of calcite from ACC. The latter one is lacking this librational vibration as it is an amorphous material, and therefore, no lattice vibration can be detected. For the generation of calcite Raman maps, the calcite librational vibration is integrated from 220-320 cm-1 and intensity color coded according to Table 5 (3.2.4). Different amounts of Mg2+ are incorporated in the exocuticle of each species and can be looked up in Table 6 (3.2.6). The thickness of the calcite layer as well as the arrangement of the calcite crystals varies in quite wide ranges due to environmental as well as vital reasons. Helleria brevicornis shows a block wise arrangement of the calcite crystals with same crystal orientation. The thickness of the exocuticle is quite homogenous (approximately 22 µm), as illustrated in Figure 23A. Corresponding average spectra are located in appendix section 7.3. The differences in the peak positions can be explained due to different Mg2+ content within the exocuticle of the different isopod species (Table 6). The thickness of the Porcellio scaber exocuticle (Figure 23B) is lower compared to other isopod species (app. 8 µm), as it

73 belongs to the group of runners and therefore a thinner exocuticle enhances the speed in running due to a lower overall mass of the cuticle. The orientation of the calcite itself is quite homogenous. Its exocuticle can be divided into a distal (thickness app. 3.5 µm) and a proximal exocuticle (thickness app. 4.5 µm), as it forms two layers lying upon each other. Although Armadillidium vulgare belongs to the group of rollers, it also has two layers like Porcellio scaber, but the thickness of the exocuticle is way higher (distal exocuticle app. 6 µm and proximal exocuticle app. 8 µm) in order to form a hard sphere in the case of predation. The Raman map for Porcellio scaber illustrating the calcite distribution is shown in Figure 23C. Another domain like arrangement of calcite crystals can be found for Tylos europaeus (Figure 23D). Big differences concerning the thickness of the exocuticle for Tylos europaeus (14-20 µm) can be detected, which can be explained due to the presence of microtubercles. The most extraordinary shape of calcite crystalline regions can be found in the marine isopod Sphaeroma serratum (Figure 23E), as calcite seems to form triangular shaped calcite domains. The diameter of the exocuticle is quite uniform having a value around 20 µm. More details on orientational properties of the calcite crystalline regions will be discussed in 4.3.3.2.

A) B) C) D) E)

8 µm 4 µm 8 µm 8 µm 12 µm

Figure 23: Raman maps showing the distribution of calcite (integration of the calcite librational vibration) in the exocuticle for 5 different isopod species using a red intensity color code. Quite different kinds of calcite formations can be found: A) Helleria brevicornis; B) Porcellio scaber; C) Armadillidium vulgare; D) Tylos europaeus; E) Sphaeroma serratum. Average spectra of the certain regions can be found in appendix section 7.3.

74

4.3.3.2 Orientational properties within the calcite crystalline region of isopod tergite cuticle systems As described in 3.2.5 the orientation of calcite can be estimated by introducing the carbonate ratio as a quantity to discriminate different orientations of calcite crystalline regions. Applying the carbonate ratio on Raman spectral maps reveals that the orientation of calcite crystalline regions within isopods can be quite different. Several arrangement types like plates, blocks as well as special forms like a triangular form can be found. In order to get an overview on the orientation of calcite within the exocuticle, it is necessary to investigate the samples from different perspectives. Therefore, for a few isopod species, samples were polished in sagittal and tangential fashion. Polarized scanning confocal microscopy was used at 0° and 90° incident laser light orientation, which corresponds to parallel and perpendicular laser light orientation with respect to the sample surface. The results shown in the next two sections (4.3.3.3 and 4.3.3.4) are already published ([41]), but shall be discussed as the measurements were performed by the author of this work.

4.3.3.3 Calcite orientation within the tergite cuticle of Porcellio scaber As a first example, the exocuticle of Porcellio scaber shall be investigated in sagittal and tangential perspective. The corresponding Raman maps are visualized in Figure 24, showing calcite, carbonate and the CR measured with 0° incident laser light orientation (parallel) in section A-C and 90° incident laser light polarisation (perpendicular) from section D-F. The calcite maps in both laser polarisation directions are just showing slight changes, whereas the carbonate maps are revealing that at least 3 areas having different calcite crystalline orientation can be estimated. Confirmation is found calculating the CR values for both measurements. A multi color code is used to show also small variations in the CR values. The three detected areas are having a plate like arrangement. As these layers are lying upon each other, they exocuticle can be divided into a distal and proximal exocuticle. A summary of the values determined for the three sagittal regions can be found in Table 11. The CR values for both areas a and b are just varying by a factor of 1.1 comparing 0° and 90° incident laser light polarization. Therefore, it can be estimated that these plates are both well aligned along the c-axis of the calcite unit cell, as their CR values

75 are quite low at 0.15 for plate a and 0.22 for plate b in average. These two regions are forming the distal exocuticle and region c is representing the proximal exocuticle. Plate c shows a noteable higher CR value of 0.71 for 0° laser light polarisation, indicating a different alignment of the calcite crystals within this layer. Comparing the CR values of plate c at 0° and 90° polarisation, a change by factor 2.8 can be observed, which is a good indication that some variations in the x-y plane of the unit cell are happening and therefore a tilt out of the plane. These results are also in good agreement to corresponding and published EBSD measurements ([41]).

A) calcite 0° pol. D) calcite 90° pol.

B) carbonate 40 µm E) carbonate 40 µm

C) carbonate ratio (CR) 40 µm F) carbonate ratio (CR) 40 µm

40 µm 40 µm G) average CR regions 0 0.8 c b

a

Figure 24: Sagitally prepared tergite cuticle sample of Porcellio scaber, investigated with polarized confocal Raman mapping using 0° (A-C) and 90° (D-F) incident laser light polarization orientation: A, D) Raman map resulting after integration of the calcite librational vibration; B, E) the carbonate stretching vibration; C, F) map showing the distribution of the calculated CR values using an intensity color code ranging from 0 to 0.8; G) Averaged regions having similar CR values.

The preparation of tangentially prepared exocuticle tergite cuticle samples is quite difficult as just the upper few micrometers are of particular interest. Therefore, a careful polishing process has to be applied as the exocuticle of Porcellio scaber is just about 5 µm in diameter. In case of Porcellio scaber, the polishing process was performed slightly too deep exposing additionally some endocuticle in the center of the polished sample and therefore also some ACC can be found. Figure 25A, D is showing that problem, but the exocuticle still can be characterized as calcite can be detected. Again, only three general regions with similar CR values can be identified, which are depicted

76 in Figure 25C,F and G. Region a again seems aligned quite well in the c-direction of the calcite unit cell, although a change of factor 1.7 can be observed changing the laser polarization by 90°. The CR values can be looked up in Table 11. For region b, a change of the factor 1.6 was calculated comparing the CR values at 0° and 90° incident laser light orientation. The CR values of 0.32 (0° pol.) and 0.52 (90° pol.) are indicating a slight switch out of the c-axis of the calcite unit cell. A more detailed look on section c reveals a similar CR ratio between 0° and 90° incident laser polarization of 1.6, but the basic CR values are way higher implying a larger tilt out of the calcite c-axis. [41]

0° pol. A) calcite B) carbonate C) CR

40 µm 40 µm 40 µm 90° pol. D) calcite E) carbonate F) CR

40 µm 40 µm 40 µm 0.8 G) average CR -regions b

a

c 0

Figure 25: Tangential prepared tergite cuticle sample of Porcellio scaber, Raman mapping using 0° (A-

C) and 90° (D-F) incident laser polarization orientation: A, D) Raman map due to integration of the calcite librational vibration; B, E) carbonate stretching vibration; C, F) distribution of the calculated CR 0.8 values using an intensity multi-color code ranging from 0 to 0.8; G) averaged regions with equal CR values.

77

Table 11: Overview of the resulting carbonate ratio values estimating the orientation of the regions as depicted in Figure 24G and Figure 25G for Porcellio scaber in sagittal and tangential perspective.

area CR (sagittal) CR (tangential)

pol. / ° 0 90 0 90

a 0.15 0.17 0.19 0.33

b 0.22 0.25 0.32 0.52

c 0.71 0.25 0.46 0.76

4.3.3.4 Calcite orientation within the tergite cuticle of Armadillidium vulgare For Armadillidium vulgare, the organization of calcite crystals is slightly different compared to Porcellio scaber, which is illustrated in Figure 26. Again Raman maps with parallel (0° pol., Figure 26A-C) and perpendicular (90° pol., Figure 26D-F) polarized incident laser light orientation were measured. The calcite crystals are less organized forming blocks with the same calcite crystal orientation. These blocks with a length of 30-50 µm are forming the distal exocuticle (Figure 26G). A small layer with uniform calcite crystal orientation is located beneath these blocks, which corresponds to the proximal exocuticle. The total thickness of the exocuticle of Armadillidium vulgare is about 10 to 15 µm, which is about 3 times higher compared to Porcellio scaber. The main reason for that can be found in the defense mechanism of Armadillidium vulgare, which rolls into a sphere in case of predation and therefore a thicker exocuticle is needed to form a hard protective shield. Porcellio scaber on the other hand belongs to the group of runners. A more lightweight construction therefore is beneficial to increase its running speed in case of predation. As already mentioned, region a in section G of Figure 26 is quite different as it is a very small layer between the block-wise arrangement (region b-f) of calcite crystals (distal exocuticle) and the endocuticle of Armadillidium vulgare, representing the proximal exocuticle. This layer with a thickness of 1-2 µm can be found along the whole thoracomere. The orientation of the calcite blocks within the distal exocuticle in general is quite different compared to Porcellio scaber. The CR values for the 6 highlighted regions in section G (Table 12) are slight higher for Armadillidium vulgare indicating a

78 larger tilt out of the c-axis of the calcite unit cell, but again the ratios between the CR of 0° and 90° polarization are around the factor 1.5.

A) calcite 0° pol. D) calcite 90° pol.

B) carbonate 80 µm E ) carbonate 80 µm

C) CR 80 µm F ) CR 80 µm

80 µm 80 µm 0 0.5 G) average CR-regions c b d e f a

Figure 26: Sagitally prepared tergite cuticle sample of Armadillidium vulgare, investigated with polarized confocal Raman mapping using 0° (A-C) and 90° (D-F) incident laser light polarization orientation: A, D) Raman map resulting after integration of the calcite librational vibration; B, E) integration of the carbonate stretching vibration; C, F) map showing the distribution of the calculated CR values using an intensity multi-color code ranging from 0 to 0.8; G) Averaged regions with equal CR values.

For confirmation, again a tangential Raman map was acquired as shown in Figure 27A- G, where 9 major areas with different kinds of shapes can be identified. The block wise arrangement of calcite crystalline regions of the distal exocuticle as already demonstrated for the sagittal prepared sample of Armadillidium vulgare also reflects in the tangential prepared sample. Compared to Porcellio scaber, much more regions with equal calcite orientation can be identified. Additionally, the size of the calcite regions with same CR value varies from 10 up to 150 µm in diameter. The CR values are way higher compared to the sagittal measurements indicating that the major orientation of the areas has to be perpendicular to the tangential face. This fact can also be confirmed by EBSD measurements as they are shown in literature ([41]). A summary of the investigated CR values concerning the tangential polished sample can be found in Table 12. Besides the orientation of the calcite regions, two pore canals can be identified, which are very important for the transport of different materials like ACC and water. ACC transport is very important for the growth of the isopod in the so named molting cycle. Herein, part of the calcite gets resorbed and is stored in the sternal ACC deposits at the lower body side of the isopod, before it is reused forming a new larger exocuticle. The

79 molting process is divided into two major stages as molting first takes place in the anterior part and then the posterior. [21]

A) calcite 0° pol. D) calcite 90° pol.

50 µm 50 µm

B) carbonate E ) carbonate

50 µm 50 µm

C) CR F) CR

50 µm 50 µm

0 0.5

G) average CR-regions

i e g h b c a f d

Figure 27: Tangential prepared sample of the tergite cuticle of Armadillidium vulgare. The sample was investigated with polarized confocal Raman mapping using 0° (A-C) and 90° (D-F) incident laser light polarization orientation: A, D) Raman map resulting after integration of the calcite librational vibration; B, E) integration of the carbonate stretching vibration; C, F) maps showing the distribution of the calculated CR values using an intensity multi-color code ranging from 0 to 0.5; G) Averaged regions having same CR values are shown. The corresponding CR values are depicted in Table 12.

80

Table 12: Overview of the resulting carbonate ratio values estimating the orientation of the regions as depicted in Figure 26 and Figure 27 for Armadillidium vulgare in sagittal and tangential prepared samples.

area CR (sagittal) CR (tangential)

pol. / ° 0 90 0 90

a 0.18 0.18 0.25 0.72

b 0.30 0.19 0.65 0.28

c 0.44 0.29 0.44 0.42

d 0.43 0.12 0.61 0.27

e 0.23 0.25 0.25 0.44

f 0.14 0.35 0.47 0.51

g 0.46 0.30

h 0.61 0.23

i 0.47 0.28

4.3.3.5 Calcite orientation within the tergite cuticle of Tylos europaeus The edges of the tergite cuticle of Tylos europaeus are of particular interest in terms of their shape and orientation of the calcite crystalline regions. Therefore, both edges of the Tylos europaeus thoracomere were investigated on their calcite crystalline orientation. As the sagittal edges were very sensitive to the incident laser radiation, just measurements at 0° incident laser polarization were performed as serious degradation of the sample occurred. The distribution of the calculated CR values as well as averaged CR regions is visualized in Figure 28. In Figure 28A, the CR of the anterior edge is shown having a quite uniform thickness around 25 µm. The shape and size of the calcite crystalline regions with equal orientation are differing in quite wide ranges, which are depicted in Figure 28C. The CR values are varying from 0.24 for region c up to 0.65 for region k. Remarkable is the area summarized as region b, which can be found within the whole exocuticle of the anterior edge having a CR value of 0.3. A detailed list of the CR values of all regions can be found in appendix 7.4 in Table 15.

81

Aside from the lunate shape of the edge (posterior part) of the Tylos europaeus thoracomere in Figure 28B, a micro tubercle was measured showing a typically Tylos europaeus block wise arrangement of calcite domains as outlined in Figure 28D. Remarkable are the very thin sections of area a and b having a length of 60 and 90 µm, which are located in the lower part, reaching the final edge in the small tip. The CR values are ranging from 0.19 for area p up to 0.65 for area q. One big exception is area b having a CR value of 0.91, indicating that the calcite crystals are tilted quite far out of their c-axis in this sagittal perspective. Region a has a CR value of 0.49, which still is tilted out of the c-axis of the calcite lattice but not as much as region b.

A) CR anterior edge B) CR posterior edge

30 µm 20 µm

0.5 0

C) average CR-regions anterior edge D) average CR-regions posterior edge m n m k l b o l h i r q p g j k j d i e b h c f g a f d e c 30 µm a 20 µm b

Figure 28: The carbonate ratio and averaged CR regions for the anterior and posterior edge of Tylos europaeus at parallel incident laser light orientation. A multi-color color code ranging from 0 to 0.5 was chosen for CR map visualization: A) CR for the anterior thoracomere edge; B) CR for the posterior thoracomere edge; C) averaged CR-regions of the anterior edge; D) averaged CR-regions of the posterior edge. Calculated CR values can be found in appendix 7.4 within Table 15.

For the tangential oriented sample, characteristic calcite crystalline regions can be identified in both parallel and cross polarized laser light fashion, which is shown in

82

0° pol. A) calcite B) carbonate C) CR

10 µm 10 µm 10 µm

90° pol. D) calcite E) carbonate F) CR

10 µm 10 µm 10 µm

G) average CR-regions b d f e c 0.5 a h g

i j k v

p

o q l u 0 t m r n s Figure 29: Tangential tergite cuticle sample of Tylos europaeus. Samples were investigated with polarized confocal Raman mapping using 0° (A-C) and 90° (D-F) incident laser light polarization: A, D) map after integration of the calcite libr. vibr.; B, E) integration of the carbonate stretching vibr.; C, F) distribution of the calculated CR values ranging from 0 to 0.5; G) Averaged regions having equal CR values. Figure 29A-F. The calcite and carbonate Raman map acquired at 0° and 90° laser light orientation with respect to the sample surface (Figure 29A, B, D and E) are showing the characteristic complementary character. The calculated CR-value maps (Figure 29C, F)

83 are in quite good agreement as the major CR regions can be compared. No preferred geometry of the calcite crystalline regions in the tangential point of view can be identified, but compared to Armadillidium vulgare the calcite crystalline regions with equal CR are much smaller in their diameter. The average ratio between CR (0° pol.) and CR (90° pol) is varying from 1 to 3.2 indicating a quite inhomogeneous distribution of the total orientation of the different CR averaged areas. For confirmation, a detailed list of the CR values is available in Table 15 in the appendix section 7.4. These results are all in good agreement to literature [17], although the investigations were performed in the central region of the tergite cuticle of Tylos europaeus there. Major modifications differing from the central region in terms of calcite orientation could be identified (i.e. the two large sections within the posterior edge of the tergite cuticle). The reason for that can be assigned to different mechanical requirements at the edge.

4.3.3.6 Calcite orientation within the tergite cuticle of Helleria brevicornis The highest thickness for the exocuticle of all investigated species was found for Helleria brevicornis having a diameter around 30 µm. In the case of Helleria brevicornis no samples in tangential fashion could be obtained and therefore just measurements in sagittal fashion could be performed. In order to have a full data set, again Raman maps at 0° and 90° incident laser light orientation were measured and the typical vibrations for calcite and carbonate integrated, which are visualized in Figure 30A-F, where additionally the carbonate ratio at both polarization angles is shown. The calcite domains formed by Helleria brevicornis are quite larger compared to the previous investigated isopod species. Furthermore, no preferred shape for the domains can be detected. The values for the average CR-values as shown in section G are quite low between 0.2 and 0.4 with just a few exclusions, indicating a quite good alignment of the calcite crystals with respect to the c-axis of the calcite unit cell. Compared to the sagittal CR Raman maps of Armadillidium vulgare and Porcellio scaber, no discrimination into distal and proximal exocuticle can be made. In total, 30 different CR-regions could be identified. The full list of all areas is located in appendix section 7.5 in Table 16.

84

A) calcite 0° Pol. D) calcite 90° Pol.

50 µm 50 µm B) carbonate E) carbonate

50 µm 50 µm C) CR F) CR

50 µm 50 µm 0 0.5

G) average CR-regions ad o q s u x h j l aa a c e p d f g i m n t v w y ab b k r z ac

Figure 30: Sagitally prepared sample of Helleria brevicornis. The sample was investigated with polarized confocal Raman mapping using 0° (A-C) and 90° (D-F) incident laser light polarization angles: A, D) Raman map integrating the calcite librational vibration; B, E) the carbonate stretching vibration; C, F) distribution of the calculated CR values using an intensity multi-color code ranging from 0 to 0.5; G) Averaged regions having equal CR values. Table 16 in section 7.5 is listing all calculated CR values.

4.3.3.7 The exocuticle of the marine isopod Sphaeroma serratum In order to have a contrast to all the terrestrial isopods, the exocuticle of the marine isopod Sphaeroma serratum was investigated in terms of its calcite orientation as well. As Sphaeroma serratum is a sphere forming isopod like most of the other investigated species, its exocuticle has a diameter around 30 µm but is slightly smaller in its diameter compared to Helleria brevicornis. The calcite orientational properties differ from the previous examined specimen, as there just seem to be 2 major preferred calcite crystalline orientations (Figure 31A-F). The identified regions are outlined in Figure 31G. Exceptionally, it seems that Sphaeroma serratum is forming triangular shaped areas (just 2 major crystal orientations), which could not be found in any of the other investigated isopod species so far. The CR values indicate that the two areas are having quite diverse orientation (Table 13) and that there is a notable tilt out of the c-axis of the calcite crystal lattice as the CR values of 0.4 for area a respective 0.3 for area b (both

85 determined using 0° incident laser light polarization) indicate. The ratio calculated out of the CR for 0° and 90° incident laser polarization for each region additionally shows a quite diverse orientation of these two areas to each other as values of 1.32 (0° pol.) and 2.9 (90° pol.) were calculated.

A) calcite 0° Pol. D) calcite 90° Pol.

50 µm 50 µm B) carbonate E) carbonate

50 µm 50 µm C) CR F) CR

50 µm 50 µm

0 0.7 G) average CR-regions a b

Figure 31: Sagitally prepared sample of Sphaeroma serratum: The sample was investigated with polarized confocal Raman mapping using 0° (A-C) and 90° (D-F) incident laser polarization orientation: A, D) Raman map resulting after integration of the calcite librational vibration; B, E) integration of the carbonate stretching vibration; C, F) map showing the distribution of the calculated CR values using an intensity multi-color code ranging from 0 to 0.7; G) Averaged regions having equal CR values.

Table 13: Carbonate ratio values estimating the orientation of the regions as depicted in Figure 31.

area CR (sagittal)

pol. / ° 0 90

a 0.40 0.53

b 0.30 0.87

4.3.3.8 ACC and phosphate within the endocuticle of the isopod tergite cuticle

The chitin protein framework within the endocuticle as discussed in 4.3.2.2 is reinforced with protein stabilized ACC. Its function therefore is to fill the cavities left over by the

86 helicoidally arranged chitin protein fibers. But ACC is also very important in the molting process, when the old exocuticle gets replaced by a larger new exocuticle. Herein ACC functions as a precursor before it crystallizes in the exocuticle again. [21] But as mentioned in 3.2.4, one has to be careful in the discrimination of calcite and ACC as both are forms of calcium carbonate. Calcite and ACC are exhibiting a carbonate stretching vibration and therefore both of them are shown when the carbonate stretching vibration (1050-1130 cm-1) is integrated in terms of a Raman spectral map. But both materials easily can be discriminated as ACC is lacking the typical calcite lattice vibrations in the range from 120 cm-1 to 320 cm-1, which are replaced by a broad peak feature as discussed in 3.2.4. An orange intensity color code was chosen for the visualization of the Raman spectral maps, which are shown in Figure 32. There are big differences in the peak intensity of the carbonate stretching vibration comparing calcite and ACC, which is for calcite up to 10 times higher compared to ACC. If both materials are present in a spectral map, the brightest parts are originating from calcite. Usually it is quite easy to discriminate ACC and calcite, when the maps of the carbonate stretching vibration and the calcite librational vibration (Figure 23) are compared. The darker parts in the endocuticle can be referred to ACC. As a conclusion of this, ACC can be detected within the whole endocuticle of all 5 investigated isopod specimens. Calculated average spectra (appendix section 7.1) are showing that at least three different components can be identified within the endocuticle of all 5 investigated isopod specimens. Besides ACC, also chitin protein fibers (4.3.2.2) and phosphate can be found. Further methods in the discrimination of calcite and ACC, such as peak shifts to lower wavenumbers and the increase of the FWHM of the carbonate stretching vibration due to the higher amorphous character of ACC shall not be discussed again and can be looked up in Figure 10A and B (section 3.2.4). The organization of ACC is quite homogenous for all investigated isopod species and can be detected in the whole endocuticle as depicted in Figure 32. This is quite plausible as the main function of ACC is the support of the chitin protein fibers.

87

A) B) C) D) E)

8 µm 4 µm 8 µm 8 µm 12 µm

Figure 32: Raman maps showing the distribution of carbonate (integration of the carbonate stretching vibration) for 5 different isopod species using an orange intensity color code: A) Helleria brevicornis; B) Porcellio scaber; C) Armadillidium vulgare; D) Tylos europaeus; E) Sphaeroma serratum.

The function of phosphate within the endocuticle is still not fully understood because some isopod species are lacking of phosphate as already described in 4.2. A different question is the origin of phosphate, which could be either of organic or inorganic origin. Raman spectral imaging reveals the distribution of phosphate, which is restricted to the endocuticle of the tergite cuticle. Within four of the five investigated isopod species, phosphate could be detected as illustrated in Figure 33. Just in the case of the marine isopod Sphaeroma serratum, it was not possible to identify any phosphate, which is in good accordance to recent literature ([15, 16]) but does not clarify why Sphaeroma serratum is lacking phosphate and why it does not need any phosphate. Furthermore, the distribution of the phosphate within the endocuticle of the four species is quite different. In Helleria brevicornis, phosphate can be detected within the whole endocuticle with an increased phosphate content in the upper half of the endocuticle (Figure 33A). This is also the case for Porcellio scaber (Figure 33B). For Armadillidium vulgare (Figure 33C) phosphate organization is slightly different as increased phosphate content can be identified in the upper third of the endocuticle. In the case of Tylos europaeus (Figure 33D) a layered arrangement of phosphate can be revealed. Average spectra of the just mentioned areas were determined and can be found in appendix section 7.6.

88

A) B) C) D)

8 µm 4 µm 8 µm 8 µm

Figure 33: Raman maps showing the distribution of phosphate (integration of the phosphate stretching vibration) for 4 different isopod species using a cyan color code: A) Helleria brevicornis; B) Porcellio scaber; C) Armadillidium vulgare; D) Tylos europaeus; Average spectra of the bright parts were calculated and can be looked up in appendix 7.6. For Sphaeroma serratum, it was not possible to detect phosphate.

4.3.4 Deviations from classical tergite cuticle structuring / the properties of thoracomere edges As mentioned in 4.2 the tergite cuticle of isopods consists of a smooth anterior part (Figure 34A) and a rough surfaced posterior part (Figure 34F), where antenna and sensilla are attached and microtubercles are integrated within the exocuticle. The big difference in the appearance of anterior and posterior thoracomere part can be described by the different function of these two parts, because the anterior part of the tergite cuticle is supposed to slide below the posterior part of the prior thoracomere. Just in case of danger, the anterior part is important when the isopod is forming a sphere in order to cover the whole inner side of the body. A full sketch of a thoracomere showing anterior and posterior part can be found in Figure 14C in section 4.2. [17] As the two main thoracomere parts are differing in their appearance, also a detailed examination of the edges is worthwhile. A look on the microscope images in Figure 34A, F reveals a quite diverse appearance of the edges. The main reason for the different shape can be assigned to the different required mechanical properties at both edges. This is the main reason for a change of the spatial distribution of the classical tergite cuticle components. The calcite spectral map (Figure 34B) of the anterior edge reveals that the calcitic exocuticle does not last until the final end of the anterior part.

89

Big differences in peak intensity within the calcite region can be detected, which can be explained by the different calcite crystalline orientations, which has been already discussed in 4.3.3.5. The structure of the endocuticle is classical despite of the change in the orientation towards the edge of the tip, exhibiting a slight curvature (Figure 34C-E). Additionally, section D shows the waxy epicuticle and membranous layer (consisting solely out of chitin protein fibers). Remarkable is the high organic content at the end of the tergite cuticle, where a high content of chitin can be detected. The high organic content at the tip might be necessary as high flexibility is needed there when the isopod is forming a sphere. Phosphate is present within the whole endocuticle, but higher concentrations are found near the edge of the cuticle and in the transitional region from the endocuticle to the membranous layer. In the center of the endocuticle, phosphate is having a layered arrangement following the given curvature of the chitin protein fibers. The posterior edge shows much more features compared to the anterior edge. Besides the lunate shape, other typical features of the posterior part like microtubercles consisting out of calcite can be identified as depicted in Figure 34G. More details on the calcite orientation of the tergite cuticle edges of Tylos europaeus and the microtubercles can be found in section 4.3.3.5. The thickness of the exocuticle decreases down to 2 µm approaching the final end of the tip. Interestingly, ACC cannot be found until the end of the tip as well as phosphate. These components seem to be restricted from the final end of the posterior edge, approximately where the diameter of the tip is becoming smaller than 15 µm (Figure 34H, J). The chitin protein fibers are following the lunate shape of the edge and the stacking height of these fibers is decreasing more and more approaching the tip of the posterior edge, which is visualized in Figure 34I. Furthermore, the waxy epicuticle is pronounced quite well surrounding the whole exocuticle until the end of the tip. Besides calcite, organic material is the main component at the tips as the Raman map of the organic material visualizes, which is necessary in order to keep the tip flexible for movement. It seems that orientation is not that important in the tip of the posterior edge (Figure 34H, I), as no changes, either in the exo- or endocuticle could be identified. The orientation of calcite has been already discussed in 4.3.3.5.

90

A) anterior edge F) posterior edge

100 µm 30 µm

B) calcite G) calcite

30 µm 10 µm

C) carbonate H) carbonate

30 µm 10 µm

D) organic I) organic

30 µm 10 µm

E) phosphate J) phosphate

30 µm 10 µm Figure 34: The anterior (A-E) and posterior (F-J) edge of the tergite cuticle of Tylos europaeus: A, F) microscope image of the anterior and posterior edge, Raman maps are representing several components of the corresponding edge.; B, G) integration of the calcite librational vibration; C, H) integration of the carbonate stretching vibration; D, I) organic (integration of the C-H stretching vibration); E, J) integration of the phosphate stretching vibration.

91

4.4 The joint head cuticle of Porcellio scaber, Tylos europaeus and Helleria brevicornis

An example to show deviations from classical hierarchical cuticle arrangement is the joint head cuticle and its transition to the arthrodial membrane, demonstrated on the species of Porcellio scaber [48], Tylos europaeus and Helleria brevicornis. The joint head belongs to the basis of the walking legs, labelled pereiopod (Figure 35A). In combination with a socket like structure, the connection between the leg and the main body is formed where the coxal plate is located. The socket and the joint head are connected via a thin arthrodial membrane to achieve a proper movement of the leg. The shape of the joint head is more complex compared to tergite cuticle as described in 4.2, as it is distorted by 90°. A cut through the joint head cuticle marked with a white arrow within Figure 35A reveals the joint head cuticle, which has a horse shoe like appearance as shown in Figure 35B. Comparing the inner and outer edge reveals a varying thickness of the joint head cuticle. The radius of the horse shoe increases, if the cut is performed alongside the joint head Figure 35C. [48] As the joint head is an important component for the movement of the isopod, also different mechanical requirements apply. Therefore, adaptions have to be made in the construction as well as the chemical composition in order to meet the necessary requirements, which is also very interesting in terms of biomimetic applications. Although the classical elements like epi- endo- and exocuticle can still be identified, the elemental distribution is slightly different, which will be shown in the next section. [48]

Figure 35: SEM-images showing an overview of the joint head cuticle of Porcellio scaber; A) location of the joint head; B) the inner and outer side of the joint head, additionally a cross section through broadside of the joint head showing horse shoe like character; C: A cut along the long axis of the joint head. [48]

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For a detailed investigation of the elemental distribution, EDX was performed, which allows the characterization of the distribution of magnesium, calcium as well as phosphorous. The structure was investigated by SEM. [48] In this work the focus is on the distribution and arrangement of the various chemical components using confocal Raman microscopy. Due to the fine geometry of the joint head, it was very difficult to perform a proper Raman mapping of the whole joint head in a single Raman mapping sequence, as degradation of the sample occurred after some time during the measurement. This problem occurred, although the laser intensity was kept very low at 5 mW (0.5 s integration time). The degradation manifested in serious sample burning causing fluorescence as well as the 2 broad characteristic peaks for amorphous carbon (1350 cm-1 respective 1550 cm-1). Therefore, the three most important regions, both edges and a representive part in the central region were mapped separately avoiding these degradation problems. First, a sample of the joint head cuticle was prepared along the broadside of the joint head (sketch in Figure 35A). This sample was used for Raman spectral mapping. The bands for calcite, carbonate, organic matter and phosphate (references in 3.1.1) were integrated and plotted as an overlay in Figure 36A-D. The thickness of the calcite layer within the central region is slightly varying from 7-10 µm, forming two different structural elements (marked in Figure 36A, B). There is the 1- 2 µm thick outer distal exocuticle surrounding the joint head up to the edges (marked as region 1o, 1c, 1i), which does not continue onto the arthrodial membrane. The adjacent proximal exocuticle is following with a varying diameter of 4-6 µm (region 2o, 2c, 2i). The distal exocuticle is completely devoid of ACC, whereas in the proximal exocuticle some ACC can be detected. The endocuticle (4c) contains ACC, chitin protein matrix and phosphate. The latter one is showing a very weak signal almost near the detection limit in the central region. The membranous layer (5o, c) solely is made of chitin protein matrix. Insular regions containing calcite can be identified at both edges (region 3o, 3i). Approaching both edges near to the arthrodial membrane, calcite cannot be detected anymore. Instead of calcite, ACC and a high content of organic material is present, which is needed to keep the joint head flexible at the edges (6o, 6i). Furthermore, the phosphate stretching vibration is 3 times higher (7o, 7i) indicating a higher phosphate concentration as well. Phosphate can be found within the whole edge. Average spectra of 7o, i and 5o, c within the edge can be found in appendix section 7.7.

93

A) calcite B) carbonate 1o

3o 2o 1o 2o 3o outer edge 4o

2c 2c

central region 1c 4c 1c

2i 3i inner edge 3i

30 µm 1i 30 µm 1i C) organic D) phosphate

5o 4o 4o 6o 7o 5c 4c 4c

7i 6i

30 µm 30 µm

Figure 36 : The joint head cuticle of Porcellio scaber as shown in Figure 35B: Each section consists out of 3 Raman maps; Following peaks are integrated showing different components A) integration of the calcite librational vibration; B) integration of the carbonate stretching vibration; C) C-H stretching vibration, D) phosphate stretching vibration; The major structural elements are highlighted and several areas are marked with consecutive numbers and their position (outer edge = o, central region= c, inner edge = i). 1) distal exocuticle, 2) proximal exocuticle, 3) insular calcite regions within the endocuticle at both edges, additionally ACC and chitin protein matrix can be detected, 4) endocuticle, 5) membranous layer, 6) high organic content at both edges, 7) high phosphate content at both edges.

As the calcite orientation is of great importance in terms of stability of the joint head cuticle, CR- maps were generated using 0° and 90° oriented incident laser light (Figure 37A,B). 5 different areas with equal calcite orientation could be identified (Figure 37C). The distal exocuticle shows an equal CR value for the whole section with values of 0.22 (0° laser light orientation) and 0.44 for 90° incident laser light orientation, which is highlighted as region a within Figure 37C, indicating a significant tilt out of the calcite c-axis. The CR values of the four regions within the proximal exocuticle vary between

94

0.12 and 0.44. A full list of all CR values can be found in Table 14. Remarkable is region c with CR values of 0.27 (0° polarization) compared to 0.29 for 90° incident laser light orientation. Region d and e are belonging to the aforementioned insular calcite regions within the endocuticle in the outer edge. The spectra of the 5 regions at 90° incident laser light orientation are outlined in appendix section 7.7. [48]

A) CR (pol. 0°) B) CR (pol. 90°)

calcite insular region within the endocuticle

10 µm 10 µm

0 0.7 a C) average CR-regions b c

d

e e Figure 37: The outer edge of the joint head cuticle. For calcite orientation determination, CR maps at 0° and 90° incident laser light polarization were calculated: A) CR at 0° and B) CR at 90° incident laser light orientation; C) sections having same calcite crystalline orientation according to their CR value. [48]

Table 14: CR-values for a sagitally polished joint head cuticle sample of Porcellio scaber. Polarized Raman mapping was performed at 0° and 90° incident laser light polarization. The CR-values at each polarization and area were determined.

area CR (sagittal)

pol. / ° 0 90 pol. / ° 0 90

a 0.22 0.44 d 0.29 0.44

b 0.32 0.12 e 0.30 0.23

c 0.27 0.29

For a fully characterization of the joint head cuticle of Porcellio scaber, it is necessary to investigate a sample prepared alongside the joint head (sketch in Figure 35C) too. The sample reveals horse shoe like character again, but the total width is increasing to

95

220 µm. The results are shown in Figure 38 and the most interesting regions are marked with numbers in this illustration. Insular regions of calcite (1) within the endocuticle at the edge can be identified again as well as the distal and proximal exocuticle solely consisting out of calcite (Figure 38B 2), although the Raman map does not show that high peak intensity compared to Figure 36.

A) microscope image B) calcite 2 to coxal central region plates

1

outer edge inner edge 60 µm 60 µm C) carbonate D) organic

3 4

60 µm 60 µm E) phosphate F) CR-0° pol.

3

60 µm 60 µm

0 0.5

Figure 38: The joint head cuticle of Porcellio scaber prepared at the long side as shown in Figure 35B: A) microscope image of the joint head cuticle showing the connection up to the coxal plate; B) integration of the calcite librational vibration; C) integration of the carbonate stretching vibration; D) C-H stretching vibration showing the distribution of organic matter within the epicuticle, endocuticle and membranous layer; E) phosphate stretching vibration; F) CR-map at parallel laser light orientation using a multi-color code from 0 to 0.5. Important regions are highlighted. 1) insular calcite region within the endocuticle at the edge, 2) distal and proximal exocuticle, 3) wave-like character near the inner edge, 4) membranous layer.

96

ACC can be detected within the whole endocuticle (Figure 38C), showing a quite remarkable wave-like character (3) in the inner edge until the transition into the central region. The membranous layer on the inner side of the cuticle is pronounced quite well in section D (4) together with the epicuticle on the outer side of the joint head cuticle. Additionally, a high organic content can be found in the final end of the outer edge. Phosphate can be detected in the whole endocuticle up to the final end of the outer edge, although the signal is quite weak. The average CR ratio is around 0.29.

4.4.1 The joint head cuticle of Helleria brevicornis and Tylos europaeus Deviations in the structure and chemical distribution compared to Porcellio scaber can be found investigating the joint head cuticle of Helleria brevicornis and Tylos europaeus. Raman maps were obtained for both species, which are depicted in Figure 39 and Figure 40 (average spectra in appendix section 7.8, 7.9). The overall diameter of the joint head cuticle for Helleria brevicornis varies from 40 to 70 µm and 60-90 µm for Tylos europaeus. The thickness of the exocuticle of Helleria brevicornis (Figure 39A, 1o, 1c, 1i) varies from 10 to 12 µm, whereas for Tylos europaeus (Figure 40A 1o, 1c, and 1i) a variation from 15-18 µm in the thickness of the calcite layer can be observed. Contrary to Porcellio scaber, no insular calcite regions within the endocuticle at the edges can be found in the case of Helleria brevicornis and Tylos europaeus. But all three species have in common that the calcite layer is not continuing until the final end of the edges. The orientation for the calcite layer is quite homogenous for Helleria brevicornis, whereas for Tylos europaeus the characteristic calcite domains can be detected as already shown for the tergite cuticle in 4.3.3.5. Furthermore, no discrimination between distal and proximal exocuticle is possible, as for both species just a single layer (Helleria brevicornis, (Figure 39A 1o, 1c, 1i)) or domain like arrangement of calcite orientations (Tylos europaeus, (Figure 40A 1o, 1c, 1i)) could be identified. The endocuticle for both specimens contains ACC (Figure 39B and Figure 40B) as well as organic chitin protein matrix and phosphate (section C and D of Figure 39 and Figure 40 (region 2o, 2c, 2i in each case). The Raman map of organic material shows the waxy epicuticle (region 3o, 3c, 3i in Figure 39C and Figure 40C). Contrary to the joint head cuticle of Porcellio scaber, in the case of Helleria brevicornis and Tylos europaeus appendages consisting out of organic material can be found most distally in

97 addition to waxy epicuticle, which seems to form antenna out of organic material. Pretty similar is the high organic content at the inner and outer edge marked as region 4o and 4i, which can be found for both, Helleria brevicornis and Tylos europaeus. The membranous layer (5o, 5c, 5i) surrounds the proximal side of the joint head cuticle.

A) calcite B) carbonate 1o 1o

2o outer edge 1c central 2c 1c region

1i 1i 2i inner edge

30 µm 30 µm

3o C) organic material D) phosphate

2o 4o 6o

2c 5o 3c 5c 2c

5i

3i 2i 6i

4i 30 µm 30 µm

Figure 39: Raman maps showing the joint head cuticle on the broadside (short part) of Helleria brevicornis. Following peaks are integrated showing different components: A) integration of the calcite librational vibration; B) integration of the carbonate stretching vibration; C) organic chitin protein matrix (C-H vibration); D) phosphate stretching vibration; The major structural elements are highlighted and several areas are labelled with consecutive numbers and their position (outer edge = o, central region= c, inner edge = i); 1) exocuticle, 2) endocuticle showing curved arrangement, 3) epicuticle and antenna, 4) organic features at both edges ensuring flexibility, 5) membranous layer surrounding the inner side of the joint head cuticle, 6) high phosphate region at both edges. Average spectra can be found in appendix 7.8.

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The phosphate distribution within the joint head cuticle is similar to Porcellio scaber. Typically it can be found in the endocuticle. Within the central region of the joint head cuticle the distribution is quite homogenous (region 2c in Figure 39D and Figure 40D). Moving on to the edges (6o and 6i), a 3 times higher phosphate compared to the central region (2c) signal can be detected for both specimen, Helleria brevicornis and Tylos europaeus.

A) calcite B) 1o carbonate 1o 2o outer edge

central 1c 1c region 2c

inner edge 2i 1i 60 µm 1i 60 µm

C) organic D) phosphate

4o 2o 6o

5o 5c 3 2c 2c 5i

2i 6i 4i 3i 60 µm 60 µm

Figure 40: Raman maps showing the joint head cuticle on the broadside (short part) of Tylos europaeus, Following peaks are integrated showing different components: A) integration of the calcite librational vibration; B) integration of the carbonate stretching vibration; C) organic chitin protein matrix (C-H vibration); D) phosphate stretching vibration; Several interesting regions within the Raman maps are marked and labelled via ongoing numbers; 1) exocuticle, 2) endocuticle showing curved arrangement, 3) epicuticle and antenna, 4) organic features at both edges ensuring flexibility, 5) membranous layer surrounding the inner side of the joint head cuticle, 6) high phosphate region at both edges. Average spectra of the main sections can be found in appendix section 7.9.

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4.4.2 The coxal plates of Helleria brevicornis and Tylos europaeus

The coxal plate is located on the lateral side of the isopod body, where it is connected to the joint head via the arthrodial membrane. Therefore, the composition of the coxal plate is of particular interest as it belongs to the moving apparatus of the isopod as well. [48] The coxal plates of Helleria brevicornis and Tylos europaeus were investigated using Raman spectral mapping (Figure 41). Besides of the different shape, the coxal plates of both specimens are lacking of calcite and ACC is the dominant component. For Helleria brevicornis, two major regions can be identified as illustrated in Figure 41A-C. Region 1 within Figure 41A shows higher ACC content compared to the lower located region 2. Therefore, average spectra for both regions were calculated, which are shown in appendix section 7.10. Herein, the peak intensity of the carbonate stretching vibration of region 1 is by factor 2.25 larger compared to region 2, indicating a higher ACC content in region 1. The distribution of organic material is quite homogenous over the whole coxal plate for Helleria brevicornis as Figure 41B demonstrates. Just in the upper half on the outer side of the coxal plate (highlighted as region 3) a significant higher organic content can be detected. Phosphate seems limited to the upper half of the coxal plate as region 4 in Figure 41C visualizes. The coxal plate of Tylos europaeus has an elliptic shape. Again, Raman spectral maps were recorded, which can be looked up in Figure 41D-F. Herein, several interesting regions are marked and shall be discussed now. Completely different is the distribution of ACC for Tylos europaeus compared to Helleria brevicornis (Figure 41D), where ACC can be found within the whole coxal plate having quite homogenous distribution all over the whole sample. A high organic content can be found surrounding the coxal plate (region 6 in Figure 41E) and interestingly, there is a region in the center of the coxal plate, where the organic content is significantly lower (region 5). The same region additionally is lacking phosphate, which apart from that can be found in in the whole coxal plate, although the peak intensity is quite low. Just a single section in Figure 41F (region 4) has a significant higher phosphate signal (factor of 2.3) compared to the phosphate signal in the rest of the sample. For a further validation, average spectra of some regions were calculated, which are located in appendix section 7.11.

100

A) carbonate B) organic C) phosphate 3 outer 1 side 4

inner side 2

40 µm 40 µm 40 µm

D)carbonate E) organic F) phosphate

6 5 4

40 µm 40 µm 40 µm

Figure 41: The coxal plates of Helleria brevicornis (A-C) and Tylos europaeus (D-F). A, D) integration of the carbonate stretching vibration; B, E) organic chitin protein matrix (C-H vibration); C, F) phosphate stretching vibration; Several interesting regions are marked with consecutive numbers: 1) carbonate enriched region, 2) lower carbonate content, 3) high organic content within the coxal plate, 4) phosphate enriched region, 5) low organic content, 6) high organic content surrounding the coxal plate. Average spectra of certain regions can be found in appendix section 7.10 for Helleria brevicornis and 7.11 for Tylos europaeus.

4.5 The cornea eye cuticle of Sphaeroma serratum and Ligia oceanica

The cornea cuticle of isopods has to meet requirements for mechanical stability, but also for optical functions. The isopod eye consists of repeating optical units named ommatidia. [49] Depending on the species and habitat, the total number of ommatidia can vary in quite wide ranges. In the case of Ligia oceanica, about 500 ommatidia (size varies from 40-50 µm) can be found in total forming a hexagonal array and therefore the compound eye of the isopod species. For Sphaeroma serratum 90-100 ommatidia can be found in a hexagonal arrangement, each having a size between 50 to 65 µm. [49] Ommatidia are having light sensitive retinula cells. Furthermore, these ommatidia are having a dioptric apparatus, which is made upon a crystal cone as well as cornea cuticle. The latter one enables the light passing to the sensory cells of the retina and acts as a biconvex lens herein. [49] Although there are some studies focusing on the eye itself, there is less known on the structure as well as the chemical composition of the cornea

101 cuticle of arthropods. A general overview can be found in Figure 42, where microscope images of Ligia oceanica (Figure 42A, B) and Sphaeroma serratum (Figure 42C, D) are shown. Despite of the shape and the size of the ommatidia, also the number, which is 7 times larger for Ligia oceanica is differing and therefore good evidence that sight is more important for Ligia oceanica. The general arrangement of the cornea cuticle in both species is similar, as they are both having an epi-, exo- and endocuticle. Pore canals seem to be restricted to the regions between the ommatidia. For more information concerning the structuring of the compound eyes in general, a closer look on the literature is advised. [49] As the Raman measurements within this citation [49] were performed by the author of this dissertation, a focus on the Raman measurements is made and shall be discussed more detailed in the following sections.

A) cc B) hc 200 µm 70 µm C) D) cc hc 200 µm 100 µm Figure 42: The cornea cuticle of Ligia oceanica (A, B) and Sphaeroma serratum (C, D) showing different number and shapes of the ommatidia. A, C) optical image of the head capsule and the transition into the cornea cuticle having different numbers of ommatidia, abbreviations (hc= head capsule, cc= cornea cuticle); B, D) magnification in the cornea cuticle region where the ommatidia are located.

4.5.1 The eyes of Ligia oceanica

4.5.1.1 The head capsule of Ligia oceanica The classical hierarchical structural elements as previously discussed for the tergite cuticle of isopods or the joint head cuticle of isopods like exo- and endocuticle can also be found in the cuticle of the eye as well as appertaining head capsule. The dimensions of the layers of course cannot be compared to the cuticle systems originating from the thoracomere cuticle.

The head capsule of Ligia oceanica has a curved shape as depicted in Figure 42A. For a determination of the spatial distribution of the different compounds, Raman spectral maps were acquired, which are shown in Figure 43A-D. It’s very thin exocuticle (2-

102

6 µm) consisting out of calcite can be detected over the whole map Figure 43A (region 1-3). The largest diameter of the exocuticle can be found directly at the border to the ommatidial region (region 2). An average spectrum of section 1 and 2 of Figure 43A can be found in Figure 44. Directly at the ommatidium, the diameter of the exocuticle can be even below a µm (region 3). The orientation of the calcite crystalline regions is pretty uniform but slight variations can be detected as the intensity differences in Figure 43B (region 1) indicate. CR-values were calculated having a variation from 0.14 to 0.25 at parallel orientation of the laser light and sample surface. Quite unusual is the presence of organic material in the exocuticle colocalized with calcite as shown in Figure 44 for the exocuticle. The endocuticle contains ACC and organic chitin protein matrix (Figure 43A 4) but it was not possible to localize phosphate. The diameter of the endocuticle is varying from 25 to 30 µm in the head capsule and for the ommatidial region; values up to 20 µm can be detected. Furthermore, a layer having a high concentration of chitin protein matrix-

A) calcite B) carbonate 3 3

1 2 1 2 6 4

50 µm 50 µm C) organic D) carotenoid

4 6 5 50 µm 50 µm

Figure 43: The transitional region of head capsule of Ligia oceanica to the ommatidial array having several components according to the illustrated Raman maps; A) map after integrating the calcite librational vibration; B) integration of the carbonate stretching vibration; C) organic material (C-H stretching vibration); D) carotenoids (integration of C=C vibration); Several interesting regions are marked with ongoing numbers: 1) exocuticle within the head capsule, 2) exocuticle at the transition from head capsule to cornea cuticle, 3) exocuticle at an ommatidium, 4) endocuticle, 5) membranous layer, 6) carotenoids within the endocuticle of the head capsule.

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(membranous layer, Figure 43C 5) is located in the innermost region with a diameter varying from 3 to 10 µm. The highest diameter of the chitin protein matrix again can be found directly at the transitional region between the head capsule and the ommatidial region with its corresponding average spectrum shown in Figure 44. As usual for the membranous layer, no mineral can be found as the Raman spectrum confirms. A pretty interesting region can be found within the endocuticle of the head capsule as it seems to lack ACC (Figure 43B 6), but instead carotenoids can be found as shown in Figure 43D 6. There are two prominent vibrations at 1157 cm-1 and 1522 cm-1 belonging to C-C and C=C vibration of the conjugated chain in carotene. ([50]). The peak of the C=C vibration was used to generate the map for carotenoids in Figure 43D. These carotenoids are restricted to the head capsule and cannot be detected within the endo-

exocuticle, Figure 43A 1,2

endocuticle, Figure 43B,C 4

membranous layer, Figure 43C 5

carotinoid enriched region, Figure 43D 6

Figure 44: The average Raman spectra of the exocuticle (Figure 43A 1, 2), endocuticle (Figure 43B, C 4), the membranous layer (Figure 43C 5) and the carotenoid enriched area (Figure 43D 6) in the center of the head capsule of Ligia oceanica.

104 cuticle of the ommatidial region, where ACC, phosphate and chitin protein matrix are found. Interestingly, these carotenoids are underlying photo bleaching, which means that it is just possible to detect these carotenoids for each sample just once. After the photo bleaching process, only ACC and chitin can be detected.

4.5.1.2 The cornea cuticle of Ligia oceanica The cornea cuticle of an ommatidium reveals an even more complex structuring compared to the head capsule (Figure 45). A Raman map showing the structure of 4 connected ommatida was measured. Each of the once protected by a exocuticular calcite layer, which additionally is colocalized with organic material forming a layer with a diameter between 1 and 2 µm (red section in Figure 45E in terms of the cluster analysis). The corresponding average spectra according to the cluster analysis are shown in Figure 46. Directly beneath the exocuticle, a layer having a notable phosphate concentration is localized (Figure 45D, E, grey section), which has its maximum diameter of 5 µm in the center of each ommatidium. Besides phosphate, ACC and chitin protein matrix can be detected, which is confirmed by the corresponding average spectrum in Figure 46. The ACC content is decreasing moving from the yellow to the-

A) calcite B) carbonate

C) organic 50 µm D) phosphate 50 µm

pore canals 50 µm 50 µm E) Cluster analysis red grey

yellow green blue

30 µm Figure 45: Raman maps showing several components of 4 ommatidia segments of Ligia oceanica. A) integration of the calcite librational vibration; B) integration of the carbonate stretching vibration; C) organic material (C-H vibration); D) phosphate stretching vibration; E) Cluster analysis of a Raman map of 2 ommatidia allowing an even further discrimination in the content of the present components. Average spectra of the various regions of the cluster analysis can be found in Figure 46.

105 green section, whereas on the contrary organic material is increasing as the corresponding spectra in Figure 46 confirm. The blue region is completely devoid of minerals and just contains chitin protein matrix. Its main function is to stabilize and fix the ommatidia by forming the ommatidial array. Figure 45C nicely shows the distribution of the pore canals, which are absent in the central regions of the ommatidia but can be found in the region between the ommatidia.

red cluster

grey cluster

yellow cluster

green cluster

blue cluster

Figure 46: The average spectra resulting after performing a Cluster analysis on the Raman map of the cornea cuticle of Ligia oceanica as shown in Figure 45E.

4.5.2 The eyes of Sphaeroma serratum

4.5.2.1 The head capsule of Sphaeroma serratum The head capsule of Sphaeroma serratum is less curved compared to Ligia oceanica. The overall diameter of the cuticle within the head capsule is varying from 55 to 65 µm. A Raman map (Figure 47) of the head capsule reveals the classical structural elements, as already documented for Ligia oceanica. These are exo- and endocuticle, as well as

106 the membranous layer. The most important reference spectra are visualized in appendix section 7.12. The thickness of the exocuticle varies between 10 to 15 µm in case of the head capsule (Figure 47A, B 1, 2) and decreases drastically down to 4 µm reaching the region of the cornea cuticle (Figure 47A, B 3). Remarkable is the orientation of the calcite crystals as they are having similar orientation properties (domains with triangular shape having equal orientation) comparable to the tergite cuticle of Sphaeroma serratum as demonstrated in 4.3.3.6. This phenomenon is visualized in Figure 47A, B (region 1-3) showing calcite and the corresponding carbonate map. In the endocuticle (Figure 47B, C 4), the classical components ACC and organic chitin protein matrix can be identified, but again there is no phosphate. The overall thickness increases from 35 to 45 µm approaching the ommatidial region. Then a decreasing diameter of the endocuticle can be observed. The membranous layer is the most proximal layer in the hierarchical structuring of the head capsule (Figure 47D region 5) again. As already demonstrated for Ligia oceanica, a layer in the center of the head capsule can be localized, which decreases in diameter from 25 µm down to 10 µm at the transition towards the cornea cuticle, where the carotenoid enriched layer finally stops as shown in Figure 47D (region 6).

A) calcite B) carbonate 1 3 1 3 2 2 4 C) organic 90 µm D) carotenoid 90 µm

6 4 5 90 µm 90 µm

Figure 47: The transitional region of head capsule of Sphaeroma serratum towards the ommatidial array having several components according to the illustrated integrated Raman maps.: A) integration of the calcite librational vibration; B) carbonate stretching vibration; C) organic material (C-H stretching vibration); D) carotenoid (C=C vibration); Several interesting regions are marked with consecutive numbers: 1) exocuticle within the head capsule, 2) exocuticle at the transition from head capsule to cornea cuticle, 3) exocuticle at an ommatidium, 4) endocuticle, 5) membranous layer, 6) carotenoids within the endocuticle of the head capsule.

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4.5.2.2 The cornea cuticle of Sphaeroma serratum

The shape of the ommatidia of Sphaeroma serratum is a quite different, as it is more dome shaped compared to the analogue of Ligia oceanica. In order to get an overview of an array of ommatidia, a Raman map including 11 ommatidia was recorded. Plots of the various components can be found in Figure 48A-C. Again, there is a very thin protective calcite layer surrounding the ommatidia, which additionally is colocalized with some organic material. The distribution of ACC seems quite different as there are 4 ommatidia in the center of the map, which significantly are having a lower ACC content but equal organic content. A possible explanation for this phenomenon could be that these 4 central ommatidia are having a different function in vision compared the other ommatidia. Generally, ACC functions as a precursor for calcite, which is stabilized by proteins until it is needed ([11]). The innermost layer is consisting mainly of chitin, having a supportive function for the ommatidia. A closer look on a single ommatidium reveals more details on the distribution and structuring as depicted in Figure 48D-F, which can be even more improved performing a cluster analysis as shown in section G of Figure 48. Spectra for each cluster are shown in appendix section 7.13. The most distal region of course is consisting out of calcite and organic material but no ACC can be detected, which corresponds to the red colored cluster in Figure 48G. This layer is followed by a small blue colored cluster, where ACC, organic material and small amounts of calcite are present. The central region of the ommatidia is having a layered arrangement of chitin protein fibers in combination with ACC. This cluster is having a green color code within the underlying cluster analysis. A higher organic content only could be observed within the yellow cluster in the inter-ommatidial region. However, it was not possible to detect any phosphate neither in the head capsule nor in the cornea cuticle of Sphaeroma serratum, which is in good agreement to previous discussion in 4.3.3.8 as well as literature [49]. Herein the theory is that the high Mg2+

(5.65 mole% MgCO3 within calcite) concentration prevents ACC from crystallization, whereas for Ligia oceanica phosphates seem to have these function as the Mg2+ content is significantly lower (4.07 mole% MgCO3 within calcite). [15, 49] But there are still more detailed investigations necessary in order to find a proper solution.

108

A) calcite

B) carbonate 100 µm

2

C) organic 100 µm

D) calcite E) carbonate F) organic 100 µm

20 µm 20 µm 20 µm

G) cluster analysis red blue

yellow green yellow

20 µm

Figure 48: Raman maps showing several components of 11 ommatidial segments of Sphaeroma serratum (A-C). D- G are showing a single ommatidium for a more detailed analysis of the structure and G is representing a cluster analysis. A, D) integration of the calcite librational vibration representing calcite; B, E) integration of the carbonate vibration representing ACC and calcite; C, F) integration of the C-H stretching vibration representing organic material; G) Cluster analysis of the present Raman map allowing an even further discrimination in the content of the present components; The corresponding spectra are shown in appendix section 7.13.

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5 Sea urchin tooth

The sea urchin tooth of Paracentrotus lividus is a remarkable example for regeneration as it is rebuilt continuously due to environmental reasons and therefore an excellent role model in terms of biomimetics. The species Paracentrotus lividus, also known as purple sea urchin is living in the Adriatic sea in coastal regions with maximum water depths up to 53 m. The overall diameter of a body of a grown up sea urchin is around 7 cm. For defense purposes it has long sharply pointed spines made out of calcite, which are breaking if the sea urchin is predated. [51] The sea urchin tooth is of particular interest as tooth material is produced continuously. This necessary as the sea urchin lives on the hard sea ground substrate where it grazes around feeding on algae. Due to the hard sea ground substrate, the tooth material gets grinded off at the tip and in order not to run out of tooth material, fresh one is produced.[22,23,52] This is done via the crystallization of ACC into calcite forming several different structural elements like primary and secondary plates, stone and carinar process plates but also compositional elements at different scale levels (micro- and nano-scale), as despite of calcium carbonate also Mg with different concentrations is incorporated into the calcite lattice forming Mg-calcite.[42] The reason for Mg2+ incorporation is its hardening function when its incorporated into calcite. Although lots of investigations regarding to the composition have been made, there is less known on the local distribution of the calcite elements and structures which the tooth forms at different maturation stages. Early works even described the sea urchin tooth as a single calcite crystal. Confocal Raman microscopy herein is a useful tool, as despite of the orientation properties of calcite at a high spatial resolution also an Mg2+ content can be determined as already described in 3.2.6. [53, 54] First, a principal overview of the macroscopic structure shall be given. The masticator apparatus of sea urchins is made out of 5 convoluted teeth (Figure 49A-C). These 5 teeth are joining at the tips of the teeth also named anterior end of the masticator apparatus. The posterior edge is the region were fresh tooth material is produced continuously in the so called “plumula”. The tooth itself is T-shaped and curved along its longitudinal axis (Figure 49A). A single tooth is fixed in the jaw bone (Figure 49D), which is encasing the whole tooth but enables the tooth to slide forwards when the tooth

110 is grinded off. A further function of the jaw bone is its guiding function as it forces the tooth in a distinct direction that it perfectly joins the other teeth at the tip. [53, 55, 56]

A) view from above the masticator B) tooth tips at anterior end

C) side view from the masticator D) single tooth element front/back view

Figure 49: The masticator apparatus of the sea urchin tooth of Paracentrotus lividus: A) view from above the masticator showing 5 T-shaped sea urchin teeth; B) view at the tips of the 5 teeth; C) view from aside the masticator apparatus; D) one tooth element showing the how the tooth is fixed by the help of the jaw bone on the outside and the inside of the masticator apparatus; The jaw bone functions as an encasement and guiding tool for the tooth. The lower part of the tooth is named keel.

For a more detailed examination cross sections were prepared (Figure 50). In order to get an overview of the different structural elements, a cross section in the central region was prepared, which is visualized in Figure 50A. The stone is consisting out hard fibers made of high Mg-calcite (MgCO3 content within calcite up to 24 mole%). The stone is

111 surrounded by the primary plates (pp) and secondary plates, both having a lower Mg2+ content compared to the stone. As the diameter of the fibers increases towards the keel, the fibers are named prims then. The carinar process plates (cpp) are surrounding the prims. These 5 elements are formed step by step but even when all of these elements are formed, the tooth still grows in diameter (Figure 50B). Furthermore, two different stages of the tooth formation are shown. The upper scheme is near the anterior edge, whereas the lower scheme is at a stage, where all 5 structural elements are formed. Comparing these two stages reveals, that besides of the different shape of the jaw bone, the diameter of the tooth still increases even when all elements are formed. In the following chapters a detailed investigation on the formation of the sea urchin tooth starting with cross sections near the plumula will be given. The samples were investigated with confocal Raman imaging. Step by step cross sections with higher maturation grade (in direction to the anterior end) were measured in order to get an overview of the formation of the different structural elements until the final end at the tooth tip. Additionally the content of MgCO3 was determined for each structural element as it is crucial information regarding the hardness of the particular element.

A) Cross section in central region B) different levels of the tooth

pp pp stone sp sp

cpp cpp

pr 300 µm

Figure 50: General scheme of the sea urchin tooth: A) Cross section in the central region showing 5 different structural elements. primary plates (pp), secondary plates (sp) stone, carinar process plates (cpp) and prisms (pr); B) cross section near the anterior and the posterior edge of the tooth showing different maturation stages of the tooth.

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5.1 The start of the tooth growth at the posterior edge (plumula)

For a detailed investigation of the formation of the sea urchin tooth, Raman maps at different maturation levels and cross sections as well as different perspectives were made. For a fully understanding of the tooth formation stage, it is necessary to start the investigation in the very early stages of the tooth formation, where the crystallization process starts. Therefore, investigations had to start near the plumula as shown in Figure 51. Cross sections at different maturation levels were prepared and Raman maps having a focus on the carbonate material were measured. Schematically, the sample preparation is shown within the magnification of a single tooth in Figure 51.

tooth

tooth

tooth

cross section tooth 21 tooth

direction of sample

investigation Figure 51: Overview of on the masticator apparatus from above showing the 5 convoluted teeth reaching the plumula. The central region of an intact tooth is magnified to demonstrate the sample preparation in order to investigate the start of the tooth formation.

The closest possible cross section at the posterior edge in terms of sample preparation is shown in Figure 52A. At this stage, two areas of primary plates separately from each other start to grow. The Raman map showing the intensity distribution of the calcite lattice vibration and carbonate stretching vibration (Figure 52A, B) reveals the presence

113 of calcite, but at both edges the peak intensity is lower compared to central region of these four primary plates. The peak intensity of the carbonate peak is approximately a third compared to the central region (spectra can be found in appendix section 7.14). Plotting the peak position distribution and the FWHM of the carbonate peak, a more clear image of the crystallization process can be generated (Figure 52D, E). Three more areas for average spectrum determination were chosen and color coded, which is also used in the visualization of the spectra shown in Figure 53. Comparing the peak position

A) microscope image of section 1 B) calcite map

C) carbonate map

300 µm D) carbonate peak center

blue av. red av. E) carbonate

FWHM green av.

growth directions 100 µm

50 µm

Figure 52: Cross section of the sea urchin tooth of Paracentrotus lividus at the posterior end (plumula) showing 4 primary plates in their early formation stage. A) microscope image showing the cross section; B) Raman map after integration of the calcite librational vibration; C) Raman map after integration of the carbonate stretching vibration; D) the peak position distribution of the carbonate stretching vibration and the three areas used for average spectrum acquisition, which are shown in Figure 53; E) the distribution of the FWHM of the carbonate stretching vibration and schematically the growth directions of the primary plates.

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Figure 53: The average spectra corresponding to the areas marked in Figure 52D. The red spectrum is showing a high calcite content, whereas the blue and the green spectrum are showing significant ACC content. For conformation, additionally an orange ACC spectrum is shown and its contribution to the high baseline feature to the spectra highlighted via the hatched area from 90-300 cm-1. of the carbonate stretching vibration between all three average areas, only one conclusion is valid in terms of the crystallization process. The central depicted red region has a peak position of the carbonate stretching vibration of 1088.9 cm-1 indicating calcite having an Mg2+ content of 9.55 mole% within calcite. The Mg2+ content can be calculated analogue to 3.2.6. Since the FWHM of the carbonate stretching vibration is 20.5 cm-1 and the calcite lattice vibration appears at 278 cm-1, presence of Mg-calcite is confirmed. Within the red spectrum in Figure 53, the crystallization process is not already finished, which can be seen in the broad feature from 90-300 cm-1, where also the calcite lattice vibration is located. For a better visualization of this phenomenon, a reference spectrum of ACC is plotted in Figure 53 (orange spectrum) showing the contribution of ACC (shaded area) to the increased baseline of the other spectra in the region between 90 and 300 cm-1 and the presence of

115

ACC is validated. The spectra of the blue and green area on the contrary indicate that crystallization into calcite is taking place, but due to the peak position of the carbonate stretching vibration ((blue area = 1087.06 cm-1, 0.3 mole% Mg2+), (green area = 1087.3.0 cm-1, 1.5 mole% Mg2+)) and even more the lattice vibration (blue area = 266 cm-1, green area = 265 cm-1 instead of 280 cm-1 for pure calcite), there is a residual amount of remaining ACC, which shifts the lattice vibration to lower wavenumbers. This thesis is supported by the fact that the very prominent broad peak feature in the range from 90-300 cm-1 can be detected in the spectra as well, which is very characteristic for ACC as already demonstrated. Aside from the peak positions, another important factor is the FWHM of the carbonate stretching vibration, which decreases down to 20 cm-1 for biogenic calcite. For biogenic ACC a FWHM of 32 cm-1 was calculated as discussed in 3.2.4. On the one hand, there is the FWHM for the red area in Figure 52 having a value of 20.5 cm-1, which is near the FWHM for fully crystallized calcite and on the other hand there are the FWHM´s of the blue area of 22 cm-1 and 21.8 cm-1 for the green area indicating a higher amorphous character, ergo more amorphous calcium carbonate. Similar problems have been encountered in [57, 58] in terms of the sea urchin tooth and the sea urchin larval spicule, which also support the theory of ACC crystallization into calcite. The latter one describes the crystallization process starting with just a single rhombohedral calcite crystal within a syncytium. Additional ACC is incorporated and the crystal grows according to the orientation of the first calcite single crystal. [58]

Regarding to these facts, it seems obvious that the growth of the primary plates starts with stabilized ACC forming the calcitic primary plates. The growth itself takes place in 3 spatial directions. Two of them are shown schematically in Figure 52E. There is a growth of the primary plates in their longitudinal direction and additional primary plates are formed. The third direction of course is the total growth in z direction, which cannot be shown within this illustration. The next section was prepared 200 µm deeper into the anterior direction showing 14 primary plates (Figure 54). Again Raman maps were prepared and as the majority of the material is made upon calcite, a cluster analysis allows a deeper insight into the composition of the calcitic material, which is illustrated in combination with the

116 corresponding average spectra in Figure 54. The Raman map was recorded in the central region of the primary plates in order to get a more detailed insight of these primary plates and their conformation.

25 µm

Figure 54: Cluster analysis of a Raman map with corresponding average spectra 200 µm deeper compared to section 1 as described in Figure 52. The calcite librational vibration and the carbonate stretching vibration were chosen as basis for the cluster analysis.

The red colored primary plates are nearly fully crystallized as the FWHM of the carbonate stretching vibration is having a value of 20.6 cm-1. The peak position of the carbonate stretching vibration of 1089.8 cm-1 and 281.9 cm-1 for the calcite librational vibration indicate a significant Mg2+ content of 14 mole% in calcite. Compared to a calculated Mg content of 9.5 mole% for the central region in the first section, Mg2+ therefore is incorporated continuously as the tooth matures. The thickness of each primary plate is around 4 µm. The increased FWHM of 21 cm-1 and the increased

117 baseline from 90 cm-1 to 300 cm-1 (typical for ACC presence) as already discussed in terms of Figure 53 indicate, that the crystallization into calcite is not fully completed within the blue cluster and some ACC near the detection limit can be observed. As the diameter of the primary plates in the blue cluster are having a maximum thickness of 3 µm the proposed growth of the primary plates, as already described in Figure 52E is supported. The Mg2+ content in calcite is lower with 13.5 mole% in the blue cluster. Additionally, the calcite librational vibration is shifted to 280.7 cm-1 and the carbonate stretching vibration to 1089.6 cm-1.

A third cross section of the sea urchin tooth was prepared 200 µm with respect to section 2 as just discussed. Again Raman maps showing the distribution of calcite, carbonate, the shift of the peak center of the carbonate stretching vibration as well as the FWHM of the carbonate stretching vibration were prepared as highlighted in Figure 55. The so far separated regions of primary plates start to join each other in the middle by forming bended primary plates as it can be seen in Figure 55. Another important observation is the start of the formation of the secondary plates, which can be found in Figure 55C highlighted as green marked region.

A) calcite B) carbonate

growth directions

200 µm 200 µm C) carbonate peak center D) carbonate FWHM

200 µm 200 µm

Figure 55: The distribution of calcium carbonate material within the third polished region starting from the plumula. A) integration of the calcite librational vibration; B) integration of the carbonate stretching vibration; C) the calculated peak center of the carbonate stretching vibration (1080-1092 cm-1), three interesting areas are marked showing the corresponding normalized average spectra in Figure 56; D) The calculated FWHM of the carbonate stretching vibration (18-25 cm-1).

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Normalized average spectra of the red, blue and green regions in Figure 55C were calculated and plotted in Figure 56. The spectrum of the red region (red, Figure 56) indicates an already finished crystallization process of ACC into calcite, as the calcite librational vibration can be localized at 281 cm-1 in combination with the carbonate stretching vibration at 1089.7 cm-1, which additionally shows a FWHM of 20.5 cm-1 as a third argument. The calculated Mg-content for the primary plates in this area is around 13.6 %, which is approximately in the region as shown for the red cluster in Figure 54 for cross section number 2. Far more interesting are the blue and green area in Figure 55C. The blue area is having its carbonate stretching vibration at 1088.1 cm-1, but the calcite librational vibration is very weak and shifted to lower wavenumbers indicating a significant content of ACC, as the typically feature of ACC (broad band 90-300 cm-1) can be found.

Figure 56: The average spectra from several areas within section 3 of the polished sea urchin tooth. The spectra are color coded analogue to the areas as shown in Figure 55C. Red= spectrum in the central region of the primary plates, Blue= at the edge of the primary plates (growth direction of further primary plates), Green= beginning of the secondary plate formation.

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A further argument for ACC is the FWHM of the carbonate stretching vibration having a value of 29 cm-1. In the green area, the start of the formation of the secondary plates can be observed, which are fully attached to the primary plates and show a curved character. A Gauss analysis of the corresponding average spectrum supports the fact that the formation of the secondary plates again starts with ACC, as the carbonate stretching vibration is located at 1085.1 cm-1 having a typically FWHM for ACC of 24.6 cm-1. The lattice vibrations of calcite can be detected partially, but they are shifted to lower wavenumbers even more compared to the blue spectrum. Furthermore, the broad feature (typically for ACC as shown in Figure 53) can be detected. The 4th cross section was prepared 200 µm deeper compared to the preceding cross section, revealing the consolidation of the primary plates via formation of more curved primary plates, which are shown in Figure 57. Starting at the outer primary plates, the curvature of the primary plates increases more moving to the inner primary plates until the original separated primary plate regions join each other, which is outlined by the white elliptic markers in Figure 57A, B. The central region of the primary plates is fully crystallized, but more Mg2+ was incorporated as the peak position of the carbonate

A) calcite B) carbonate

200 µm 200 µm C) carbonate peak center D) cluster analysis

200 µm 200 µm

Figure 57: The distribution of calcite crystalline material in the formation of the sea urchin tooth of Paracentrotus lividus. Level 4 is showing the consolidation of the so far separated primary plate units. A) integration of the calcite librational vibration; B) integration of the carbonate stretching vibration; C) center of the carbonate stretching vibration determined via Gauss fit (1080-1092 cm-1) Regions used for average spectrum determination are highlighted in their corresponding color, red= representive for fully crystallized Mg-calcitic material, blue= formation of the secondary plates showing detectable ACC content; D) cluster analysis showing fully crystallized (red) and partly crystallized calcium carbonate.

120 stretching vibration (1090.6 cm-1) indicates, resulting in a Mg2+ content of 18 mole% within calcite (Figure 58 red normalized spectrum, corresponds to red marked region in Figure 57C). Additionally, the calcite librational vibration is shifted to 283 cm-1, which is also a sign for a higher Mg2+ content within calcite as already described in 3.2.6. Furthermore, a progress in the formation of the secondary plates can be observed looking at the peak center shift of the carbonate stretching vibration to lower wavenumbers as depicted in Figure 57C (blue elliptically marked region). The blue marked secondary plates are showing quite a significant shift in the carbonate stretching vibration down do 1084.5 cm-1, according to the corresponding average spectrum in Figure 58 within the blue normalized spectrum. Herein, the typically increased broad baseline feature in the region from 90 cm-1 to 300 cm-1 for ACC can be found (discussed previously in terms of Figure 53). Additionally, this theory gets supported by the high FWHM of the carbonate stretching vibration having a value of 32 cm-1.

Figure 58: Two important average spectra according to the areas marked in Figure 57C, showing fully crystallized material (red spectrum) and the formation of the secondary plates (blue spectrum), indicating the presence of ACC.

121

The significant peak at 200 cm-1 within the blue spectrum of Figure 58 cannot be assigned properly as no further peaks are appearing to refer it to another component than calcite or ACC. The cluster analysis shown in Figure 57D divides the level into two major clusters, which are the fully crystallized Mg-calcite cluster highlighted in the red and the partially or even non crystallized calcium carbonate already having a certain Mg2+content in the blue spectrum. The 5th cross section allows the insight to view the start of the formation of the stone region as it is visualized in Figure 59. The formation of the stone starts after completion of the curved primary plates. This is the case when the two primary plate regions have joined each other in the center. Raman maps showing the distribution of the calcite librational vibration and the carbonate stretching vibration are illustrated in Figure 59A and B, which additionally are emphasizing a further growth of the secondary plates. The shift of the carbonate stretching vibration documents the formation of the stone region (grey region in Figure 59C) having an average peak position of 1084.9 cm-1 with no significant peak in the calcite librational vibration peak area.

A) calcite B) carbonate

200 µm 200 µm C) carbonate peak center D) cluster analysis

start of stone

200 µm formation 200 µm

Figure 59: The sea urchin tooth of Paracentrotus lividus. Raman maps showing level 5 in depth with respect to the posterior edge in the plumula. Continuation of the growth and formation of the secondary plates as well as start of the stone formation in the center of the tooth are illustrated: A) Raman map showing the integral intensity of the calcite librational vibration; B) the carbonate stretching vibration; C) The center of the carbonate stretching vibration (1080-1095 cm-1); D) Cluster analysis showing different calcite maturation levels.

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The subsequent cluster analysis confirms the formation of the stone region. Average spectra representing the certain clusters can be looked up in Figure 60. The yellow cluster marked in Figure 59D shows a peak center of the carbonate stretching vibration of 1089.5 cm-1, without having any significant calcite librational vibration. Therefore it can be assumed that ACC with a significant higher Mg2+ content is present at this stage, which is already documented in literature ([42, 57]). The red cluster represents fully crystallized calcite with its carbonate stretching vibration at 1089.9 cm-1 (14.5 mole% Mg2+ within calcite) and the calcite librational vibration can be found at 282 cm-1. The Mg2+ content in the cyan cluster is slightly smaller (carbonate stretching vibration at 1089.5 cm-1 = 12.5 mole% Mg2+ in calcite). The calcite librational vibration can be addressed at 281 cm-1.

red cluster

cyan cluster

yellow cluster

Figure 60: Average spectra corresponding to the cluster as shown in Figure 59. Three different clusters can be distinguished having fully crystallized calcite (red), partially crystallized calcite (cyan) and minimal crystallized calcite, but more ACC (yellow).

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Concluding the tooth formation, a final Raman map documenting the whole extent of the growth of the stone material shall be given, which is depicted in Figure 61A-D. The peak center distribution of the carbonate stretching vibration determined via Gauss fits reveals that there are two general regions. On the one hand there is the Mg-calcite region having an average peak center at 1090.7 cm-1, which corresponds to an Mg2+ content of 18.5 mole%, and there is the yellow region where the formation of the stone takes place having an even higher Mg2+ content. Refinement by a cluster analysis shows three general regions (Figure 61D). The corresponding average spectra are shown in Figure 62. There is the red fully crystallized Mg-calcite (carbonate stretching vibration at 1090.7 cm-1 = 18.5 mole% Mg2+) having also a characteristic calcite librational vibration at 282.6 cm-1. For the blue region, equal Mg2+ content was determined, but due to the slightly increased FWHM of 21.1 cm-1 compared to 20.8 cm-1 for the red cluster, it can be assumed that the blue cluster still has a partial amorphous character. The yellow cluster shows no sign of a calcite librational vibration, but instead the broad feature typically for ACC can be identified as described previously for Figure 53. The peak position of 1092.3 cm-1 for the carbonate vibration indicates high Mg-ACC ([12, 42]). The FWHM is calculated at 28.3 cm-1, which is also typically for ACC.

A) calcite B) carbonate

200 µm 200 µm C) carbonate peak center D) cluster analysis

200 µm 200 µm

Figure 61: The sea urchin tooth of Paracentrotus lividus. Raman maps showing level 6 in depth with respect to the posterior edge in the plumula. Continuation of the growth and formation of the secondary plates and the stone are illustrated: A) Raman map showing the integral intensity of the calcite librational vibration; B) the carbonate stretching vibration; C) The center of the carbonate stretching vibration (1080- 1095 cm-1); D) Cluster analysis showing different calcite maturation levels (spectra in Figure 62).

124

red cluster

blue cluster

yellow cluster

Figure 62: Average spectra corresponding to the cluster as shown in Figure 61. Three different clusters can be distinguished having fully crystallized calcite (red), partially crystallized calcite (cyan) and minimal crystallized calcite, but more ACC (yellow).

5.2 The center of the tooth

The central region is of interest as well as the formation of the prisms out of the so called calcite fibers and carinar process plates can be investigated. Therefore, a sample in the central region was polished, revealing all characteristic structural elements like primary plates, secondary plates, stone, fibers, prisms and carinar process plates. In this region the sea urchin tooth already has its characteristic T-like shape. For a detailed investigation it is necessary to measure the tooth at several positions. 3 areas where chosen, which are shown in Figure 63A. Section 1 is located directly around the stone, where the calcite fibers are located. These fibers are growing and increasing their diameter. Exemplarily, this is shown after plotting the carbonate stretching vibration for section 1 in Figure 63B. The diameter of these fibers is below 1 µm reaching a size of 10 µm at the bottom of the carbonate map in Figure 63B.

125

A) tooth center and Raman mapping regions B) carbonate map section 1 section 1

fiber growth direction

section 2

section 3

100 µm 70 µm Figure 63: The central region of the sea urchin tooth: A) microscope image of central region of the tooth. Marked areas are the regions chosen for Raman mapping; B) Integration of the carbonate stretching vibration of section 1 showing the increasing size of the calcite fibers converting in the so named prisms.

For a more detailed overview on the growth of the prisms, section 2 (Figure 63A) provides more information on the prism generation. The corresponding Raman maps showing the distribution of calcite as well as the determined peak center for Mg-content determination are illustrated in Figure 64. Important average spectra for stone, prisms, and carinar process plates for section 2 and 3 are visualized in appendix section 7.15. In Figure 64A-C, the ongoing growth of the prisms can be observed as diameters up to 20 µm for a single prism can be determined. Furthermore the carinar process plates can be observed (abbreviation cpp) in Figure 64A and B. A determination of the center peak position of the carbonate stretching vibration (Figure 64C) confirms previous results, as the stone due to the higher shift is having a higher Mg2+ content within its calcite lattice. The stone with its carbonate center peak position of 1091.8 cm-1 and therefore 24 mole% Mg2+ within the calcite lattice yields a value in the range of previous Mg2+ content determinations. The prisms are of lower Mg2+ content (12 mole%, peak center 1089.4 cm-1) and the carinar process plates are having a slight higher Mg2+ content (13 mole%, peak center 1089.6 cm-1) compared to the prism section. In the lowest section of the tooth (section 3 in Figure 63A and Figure 64D and E) an even further increase in the diameter of the prisms can be observed, which can reach maximum thickness up 35 µm.

126

The carinar process plates in the lowest section are resting against the prism section, which is not the case in higher regions as for instance in Figure 64A. The lower regions will be grinded off first when the sea urchin uses its teeth for the feeding process.

A) calcite B) carbonate C) carbonate peak center

cpp cpp

cpp cpp

70 µm 70 µm 70 µm

D) calcite E) carbonate

100 µm 100 µm

Figure 64: The central region of the sea urchin tooth of Paracentrotus lividus. Raman maps of section 2 and 3 according to Figure 63. A-C correspond to section 2, D, E correspond to section 3: A, D) integration of the calcite librational vibration; B, E) integration of the carbonate stretching vibration; C) distribution of the peak center of the carbonate stretching vibration determined via Gauss fits (1080- 1095 cm-1); Average spectra of stone, prisms and carinar process plates of section 2 as well as prims and carinar process plates of section 3 are shown in appendix section 7.15.

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5.3 The tip (anterior edge) of the sea urchin tooth

For an investigation of the functionality of the sea urchin tooth, the tip (anterior end) of the tooth is of particular interest, as it is the region where the tooth material is under high load due to the feeding process at the hard sea ground substrate and where the tooth material therefore gets grinded of. Samples of the tooth tip were prepared in different perspectives. One sample was prepared tangentially (alongside the tooth), as shown in Figure 65A. Several structural elements like primary plates, stone and calcite fibers are visualized. More important is the investigation of the structure of the tooth from in front of the tooth starting at the final end of the tooth (Figure 65B). Two further cross sections were prepared polishing away a few µm in order to investigate the local distribution of the calcitic material having different Mg2+ content. For the front view samples, the polishing direction is marked in Figure 65A. In the subsequent section, three different levels on the front view will be discussed in order to show the abrasion process of the different structural elements present in the tooth. For the polishing procedure steps of 200 µm between each layer have been chosen.

A) tangential view on anterior tooth edge B) front view

primary plates primary plates

stone fibers from keel stone polishing direction for front view samples 400 µm 50 µm

Figure 65: Microscopic images of the anterior edge of the sea urchin tooth of Paracentrotus lividus: A) view on a tangential prepared tooth showing the progression in abrasion of the keel towards the tip as well as the primary plates and the stone; B) view on a polished tooth sample from in front of the tooth after just polishing 20 µm from the final end of the tooth revealing just stone material and a small amount of primary plates.

For a first investigation of the tooth tip, a Raman map at the ultimate edge at the front view was collected and investigated on several compounds, as depicted in Figure 66. Section A and B of Figure 66 reveal the calcitic character of the tip even at the final end of the tooth. Just a more detailed investigation on the peak position of the carbonate

128 stretching vibration with Gauss fits (Figure 66C) and a cluster analysis (Figure 66D) reveal the compositional differences due to the different Mg2+ content at the tip of the tooth within the calcite lattice. Spectra of the cluster analysis can be found in Figure 67.

A) calcite B) carbonate

stone stone

50 µm 50 µm C) carbonate peak center D) cluster analysis

stone stone

50 µm 50 µm

Figure 66: The anterior edge of the sea urchin tooth of Paracentrotus lividus. Polishing level 1 was at 20 µm distance from the ultimate end of the tooth: A) integration of the calcite librational vibration; B) integration of the carbonate stretching vibration; C) center of the carbonate stretching vibration calculated via Gauss fits (1080-1095 cm-1) and D) cluster analysis revealing three different clusters.

The peak center distribution of the carbonate stretching vibration reveals two major regions having its center at 1091.7 cm-1 (yellow region) and 1089.2 cm-1 (blue region). A further look on the peak position of the calcite librational vibration of 286.7 cm-1 (yellow) and 282 cm-1 (blue) provide further confirmation that the difference between these two regions are mainly due to their different Mg2+ content within the calcite crystalline lattice. As a further criterion, the FWHM of 23.1 cm-1 (yellow) compared to 21.7 cm-1 (blue) of the carbonate stretching vibration can be mentioned, which again indicates a higher Mg2+ content in the yellow region. The subsequent cluster analysis generally confirms previous findings with the only difference that the previous blue region can be split into two clusters. The resulting average spectra are visualized in

129

Figure 67. All three average spectra are showing typical calcite (fully crystallized) spectra with different amounts of Mg2+ and do not show any sign of remaining ACC. The high Mg2+ content in the yellow cluster is confirmed by the peak center of the carbonate stretching vibration, which again is located at 1091.7 cm-1, resulting in an Mg2+ content of 23.5 mole% within calcite in total. The difference in the Mg2+ content between the blue and the red cluster can be explained, as first the calcite librational vibration of the blue cluster (282.8 cm-1) gets shifted to higher wavenumbers (283.6 cm-1 for the red cluster), which is typically for increasing Mg2+ content ([12]) in the calcite crystalline lattice. Furthermore, the peak shift of the carbonate stretching vibration is crucial for the Mg2+ content, as a shift from 1090.4 cm-1 for the blue cluster up to 1091.1 cm-1 for the red cluster can be observed. These peak center positions are yielding Mg2+ contents of 17 mole% (blue cluster), respective 20.5 mole% for the red cluster.

red cluster

blue cluster

yellow cluster

Figure 67: Average spectra corresponding to the cluster as shown in Figure 66. Three different clusters can be distinguished having low Mg-calcite (blue), medium Mg-calcite (red) and high Mg-calcite (yellow).

130

Therefore, it can be summarized that the material withstanding the loads at the tip longest is the calcitic material having the highest Mg2+ content within the whole tooth material. These results are in good agreement to previous works like [42, 55, 56], as their results are also concluding that high Mg-calcite is the material, which withstands the forces at the tooth tip longest. Finally, a brief discussion about the dimensions of the different clusters at the anterior edge of the first cross section shall be made. The yellow cluster containing high Mg-calcite is having a maximum width of 215 µm and a maximum height of 70 µm. The red and blue cluster are having a quite irregular shape as they are leftovers from the former existing primary plates and therefore a variation in the diameter from 2 to 15 µm can be observed. The second cross section was prepared 200 µm deeper into the tooth material. On the one hand, it shows an increase in diameter of the tooth and on the other hand, the three general elements having different Mg2+ content again can be identified, as it is depicted in Figure 68. Raman maps are showing the distribution of the calcite librational and the carbonate stretching vibration (Figure 68A, B) visualizing the increasing number of primary plates moving deeper in the tooth material starting with respect to the tip of the sea urchin tooth. At this level, the shape of the primary plates located above the high Mg-calcite region are becoming more apparent, having a maximum height of 100 µm in the center. The fact that the primary plates are more pronounced compared to the previous, more anterior level supports the fact that the high Mg-calcite withstands higher loads compared to their lower Mg2+ analogs. Therefore, the primary plates are affected way earlier in abrasion due to the feeding process. The red cluster located below the yellow cluster is a relic of the fibers origination from the keel as already previous (Figure 65A from a tangential point of view and Figure 64) in 5.2. The total height of the high-Mg calcite containing yellow cluster (Figure 68C) is not changing, but its width is increasing to 480 µm. The center peak position of the carbonate stretching vibration is located at 1092.4 cm-1 indicating a slightly higher Mg2+ content of 27 mole% compared to 23.5 mole% within the calcite crystalline lattice for the yellow cluster at the previous level. The red and the blue cluster (spectra shown in appendix 7.16) again are having a lower Mg2+ content of 21 mole% (carbonate peak center at 1091.2 cm-1) for the red cluster respective 18.5 mole% (carbonate peak center at 1090.7 cm-1) for the blue cluster.

131

A) calcite B) carbonate

100 µm 100 µm

C) cluster analysis

primary plates

stone

100 µm

Figure 68: The anterior edge of the sea urchin tooth of Paracentrotus lividus. Polishing level 2 was 200 µm deeper into the tooth material with respect to level 1: A) integration of the calcite librational vibration; B) integration of the carbonate stretching vibration; C) center of the carbonate stretching vibration calculated via Gauss fits (1080-1095 cm-1) and D) cluster analysis revealing three different clusters.

The third cross section was prepared 200 µm deeper into the tooth material with respect to level 2, which provides even more confirmation that the lower Mg2+ containing regions are grinded off before abrasion of the high magnesium calcitic stone takes place as visualized in Figure 69. In section A and B, additionally the porous character of the tooth can be seen. The total height of the yellow highlighted stone region doesn´t change at all, although the total width increases up to 600 µm. The fibrous calcite section below the yellow stone region becomes more apparent as shown in Figure 69C, D. The calculated values for the Mg2+ content (within the calcite crystalline lattice) of

132 the clusters shown in Figure 69D are 25 mole% for the yellow cluster (carbonate stretching vibration at 1092 cm-1), 16.5 mole% for the red cluster (carbonate stretching vibration at 1090.3 cm-1) and 19.5 mole% for the blue cluster (carbonate stretching vibration at 1090.3 cm-1). The corresponding spectra can be found in section 7.17.

A) calcite B) carbonate

100 µm 100 µm C) carbonate peak center D) cluster analysis

primary plates primary plates

stone stone

100 µm 100 µm

Figure 69: The anterior edge of the sea urchin tooth of Paracentrotus lividus. Polishing level 3 was 200 µm deeper into the tooth material with respect to level 2: A) integration of the calcite librational vibration; B) integration of the carbonate stretching vibration; C) center of the carbonate stretching vibration calculated via Gauss fits (1080-1095 cm-1) and D) cluster analysis revealing three different clusters. The corresponding spectra can be found in appendix section 7.17.

A tangentially prepared sample of the sea urchin tooth shall be discussed to validate the previous findings. The calcite and carbonate map as shown in Figure 70A and B are showing the three main regions present at the tooth tip. These are the primary plates, the high Mg-calcite and the calcite fibrous region below the stone region (Figure 70C, D). The peak center of the carbonate stretching vibration of the stone (yellow cluster in

133

Figure 70D) is located at 1091 cm-1 (25 mole% Mg2+) followed by 1089.3 cm-1 (11.5 mole% Mg2+) for the red cluster and 1089.2 cm-1 (11 mole% Mg2+). The calculated average spectra are shown in appendix section 7.18. The tangential Raman map therefore confirms the presences of high Mg-calcite until the final end of the tooth. Mg2+ therefore plays a major role in the function of the tooth of Paracentrotus lividus.

A) calcite B) carbonate

70 µm 70 µm C) carbonate peak center D) cluster analysis

primary plates primary plates

stone stone

calcite fibers calcite fibers

70 µm 70 µm

Figure 70: The sea urchin tooth of Paracentrotus lividus. Tangentially prepared sample showing structural elements with different Mg2+ content in the calcite crystalline lattice after Raman spectral mapping. A) integration of the calcite librational vibration; B) carbonate stretching vibration; C) peak center of the carbonate stretching vibration determined via Gauss fits (1080-1095 cm-1); D) cluster analysis showing high (yellow), medium (red) and low (blue) Mg-calcite.

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6 Conclusion

To sum up, confocal Raman microscopy is a quite versatile tool in the investigation of biomineralized systems, as it provides information on chemical and structural changes in aforementioned systems. The high spatial resolution of around 400 nm in combination with the Raman spectral mapping technique allows the investigation of highly detailed sample features. Using laser light with different polarization direction furthermore allows the investigation of changes due to orientation in the case of crystalline material (i.e. calcite), but also the twist of chitin fibers can be investigated. More advanced tools for data evaluation like Gauss fits or cluster analysis are providing even more details on the investigated sample, which could be the discrimination of calcium carbonate due to different amounts of incorporated magnesium in example. A big advantage compared to other techniques is the fact that both organic and inorganic materials can be distinguished. Crystalline sample materials with same composition but different Bravais lattice can be discriminated as well due to their characteristic lattice vibrations, as it is the case for calcite, aragonite and vaterite in example. In order to do so, also precision in terms of the spectral resolution is needed. The spectral resolution can be minimized down to 0.9 cm-1 by choosing the 1800 g/mm grating, yielding even more information on the investigated samples. It could be demonstrated that the tergite cuticle systems of isopods are adapting in size and composition due to their vital needs in terms of their defense mechanism and their environmental properties like the habitat. The classical structural elements like exo-, endocuticle and membranous layer could be identified for each species, although some properties like the stacking height of the twisted plywood like chitin protein fibers can vary in quite wide ranges. Furthermore, it could be shown that the calcitic exocuticle is having different structures due to changed orientation of the calcite crystals forming different structural elements like domains, layers and agglomerates. The endocuticle usually contains chitin protein matrix, which is supported by stabilized ACC (stabilization either with phosphate or high Mg2+ content, but still matter of scientific discussion). A quite interesting find is the difference of the phosphate content comparing the different isopod species, although it could not be clarified totally why some species are totally lacking of phosphates.

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The joint head cuticle of isopods is a good example showing deviations from classical hierarchical structuring compared to the tergite cuticle of isopods. Principally, these elements are still present, but the composition is quite different, as calcite can be found in the endocuticle at the edges of the joint head cuticle. Another difference is the high phosphate and organic material concentration at the edges of the joint head cuticle, which might be due to higher loads present at the edges on the one hand and also the higher flexibility achieved through the organic material on the other hand. A further interesting find concerns the coxal plates, which are the counter part to the joint head cuticle located on the torso of the isopod, lacking totally of calcite. The joint head cuticle is a good example for biomimetic studies, which could be quite useful for humankind in future, although just the beginning is made in these studies. The cornea cuticle and the head capsule provide useful information on the vison process of the isopods and the structure of their facet eyes. Quite diverse shapes of these facets were found and also the number of facets is differing in quite wide ranges comparing the investigated samples. Remarkable is the fact that high phosphate concentrations were found in the center of the facets of Ligia oceanica. Also compositional changes were found for facets of the same isopod species, which might be due to different requirements in vision depending on the position of the facet within the eye. The sea urchin tooth of Paracentrotus lividus is an excellent sample studying the crystallization process of ACC into calcite as tooth material has to be produced continuously due to abrasion process at the tip of the tooth. The reason for that abrasion is the hard sea ground substrate grinding off the tooth and therefore, in order not to run out of tooth material, fresh one has to be produced. It was demonstrated that the formation process starts via construction of two independent primary plate sections out of ACC. These independent sections are growing until they join in the center between these sections. Afterwards, the formation of the secondary plates and the stone starts. Later on prisms and carinar process plates are formed. The latter two elements are forming the keel, giving the tooth its unique T shape. The Mg2+ content in these sections is increasing continuously having the highest Mg2+ content in the stone region (27 mole%). The high Mg-calcite containing stone is the hardest element of the tooth material and therefore withstanding the loads at the tooth tip longer compared to the lower Mg-containing calcite elements.

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7 Appendix 7.1 Average spectra of the endocuticle (tergite cuticle of several isopod specimen)

Helleria brevicornis

Porcellio scaber

Armadillidium vulgare

Tylos europaeus

Sphaeroma serratum

Figure 71: Normalized average spectra of the endocuticle of different investigated isopod species like Helleria brevicornis, Porcellio scaber, Armadillidium vulgare, Tylos europaeus and Sphaeroma serratum as mentioned in the results and discussion part. ACC, chitin protein fibers and phosphate are dominating the average spectra determined in this region.

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7.2 Average spectra of the membranous layer (tergite cuticle of several isopod specimen)

Helleria brevicornis

Porcellio scaber

Armadillidium vulgare

Tylos europaeus

Sphaeroma serratum

Figure 72: Normalized average spectra of the membranous layer of different investigated isopod species like Helleria brevicornis, Porcellio scaber, Armadillidium vulgare, Tylos europaeus and Sphaeroma serratum as mentioned in 4.3.2.2.

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7.3 Average spectra of the exocuticle (tergite cuticle of several isopod specimen)

Helleria brevicornis

Porcellio scaber

Armadillidium vulgare

Tylos europaeus

Sphaeroma serratum

Figure 73: Normalized average spectra of the exocuticle of different investigated isopod species like Helleria brevicornis, Porcellio scaber, Armadillidium vulgare, Tylos europaeus and Sphaeroma serratum as mentioned in 4.3.3.1.

7.4 CR-values for the anterior and posterior edge of Tylos europaeus

Table 15: CR values for Tylos europaeus edges in sagittal fashion at parallel polarization and a tangential polished sample at parallel and perpendicular incident laser light polarization.

area CR (sagittal) CR (tangential)

anterior edge posterior edge

0° pol. 0° pol. 0° pol. 90° pol.

a 0.59 0.39 0.47 0.34

b 0.30 0.91 0.33 0.80

139 area CR (sagittal) CR (tangential)

anterior edge posterior edge

c 0.24 0.56 0.45 0.49

d 0.34 0.67 0.38 0.39

e 0.33 0.44 0.33 0.50

f 0.39 0.39 0.66 0.31

g 0.25 0.66 0.64 0.31

h 0.33 0.37 0.31 0.54

i 0.24 0.57 0.46 0.51

j 0.43 0.38 0.45 0.61

k 0.45 0.27 0.27 0.86

l 0.40 0.38 0.66 0.21

m 0.26 0.50 0.29 0.74

n 0.28 0.30 0.42

o 0.55 0.33 0.57

p 0.19 0.43 0.44

q 0.65 0.44 0.39

r 0.23 0.06 0.19

s 0.41 0.28

t 0.29 0.67

u 0.25 0.47

v 0.26 0.55

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7.5 CR-values for sagitally prepared exocuticle of Helleria brevicornis

Table 16: CR-values for a sagittal polished exocuticle sample of Helleria brevicornis. Polarized Raman mapping was performed at 0° and 90° incident laser light polarization. The CR-values at each polarization and area were determined.

area 0° pol. 90° pol. area 0° pol. 90° pol.

a 0.32 0.27 p 0.86 0.24

b 0.25 0.25 q 0.62 0.22

c 0.70 0.20 r 0.40 0.25

d 0.19 0.17 s 0.33 0.27

e 0.38 0.21 t 0.20 0.45

f 0.16 0.27 u 0.55 0.27

g 0.26 0.22 v 0.18 0.28

h 0.45 0.26 w 0.34 0.19

i 0.20 0.23 x 0.53 0.25

j 0.26 0.22 y 0.16 0.16

k 0.41 0.22 z 0.42 0.21

l 0.20 0.21 aa 0.47 0.27

m 0.25 0.35 ab 0.32 0.24

n 0.58 0.17 ac 0.58 0.21

o 0.25 0.24 ad 0.37 0.23

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7.6 Average spectra of the phosphate enriched parts in the endocuticle (tergite cuticle of several isopod specimen)

Helleria brevicornis

Porcellio scaber

Armadillidium vulgare

Tylos europaeus

Figure 74: Normalized average spectra of the phosphate enriched parts of different investigated isopod species like Helleria brevicornis, Porcellio scaber, Armadillidium vulgare and Tylos europaeus as mentioned in the results and discussion part.

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7.7 CR-values for sagitally prepared joint head cuticle of Porcellio scaber

area a

area b

area c

area d

area e

phosphate enriched area

membranous layer

Figure 75: The joint head cuticle of Porcellio scaber: Spectra from average regions a-e are shown at laser polarization of 90°, Furthermore, the high phosphate region in the outer edge as well as the membranous layer.

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7.8 Average spectra of the joint head cuticle of Helleria brevicornis

exocuticle

endocuticle

membranous layer

Figure 76: The joint head cuticle of Helleria brevicornis: Average spectra of classical elements are shown, which are exo-, endo-cuticle as well as membranous layer.

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7.9 Average spectra of the joint head cuticle of Tylos europaeus

exocuticle

endocuticle

membranous layer

Figure 77: The joint head cuticle of Tylos europaeus: Average spectra of classical elements are shown, which are exo-, endo-cuticle as well as membranous layer.

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7.10 Average spectra of selected regions within the coxal plate cuticle of Helleria brevicornis

Figure 78: The coxal plates of Helleria brevicornis: Calculated average spectra of the carbonate enriched region 1 (red spectrum) and the lower part of the coxal plate having lower carbonate content (blue spectrum = region 2)

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7.11 Average spectra of selected regions within the coxal plate cuticle of Tylos europaeus

carbonate region average (Figure 41D)

low organic content region (Figure 41E,5)

high phosphate region (Figure 41E,4)

Figure 79: The average spectra of three selected region within the coxal plates of Tylos europaeus: A) Average over the whole carbonate map as depicted in Figure 41D, B) Average spectrum of the small central region having low organic content in comparison to the rest of the coxal plate (Figure 41E,5), C) Average spectrum of the very small region having higher phosphate content (Figure 41F,4).

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7.12 Average spectra of the head capsule of Sphaeroma serratum

exocuticle

endocuticle

organic average

carotinoid enriched region

Figure 80: The average Raman spectra of the exocuticle, endocuticle, the organic protein matrix and the carotenoid enriched area in the center of the head capsule of Sphaeroma serratum.

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7.13 The average spectra according to the cluster analysis for the cornea cuticle of Sphaeroma serratum

red cluster

blue cluster

green cluster

yellow cluster

Figure 81: The average spectra resulting after performing a Cluster analysis of the cornea cuticle of Sphaeroma serratum as shown in Figure 48G.

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7.14 Average spectra of the central and edge region at cross section level 1 starting from the posterior edge of the sea urchin tooth of Paracentrotus lividus.

Figure 82: The average spectra of the central (black spectrum) and edge (red spectrum) at level 1 within the posterior edge of the sea urchin tooth of Paracentrotus lividus.

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7.15 Average spectra of section 2 and 3 within the central region of the sea urchin tooth.

stone section 2

prisms section 2

carinar process plates section 2

prisms section 3

carinar process plates section 3

Figure 83: Average spectra of the central region of certain areas like stone, prisms as well as carinar process plates. The spectra are originating from different sections as already depicted in Figure 63 and Figure 64.

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7.16 Average spectra according to the cluster analysis performed at level 2 at the anterior edge of the sea urchin tooth of Paracentrotus lividus.

red cluster

blue cluster

yellow cluster

Figure 84: Average spectra corresponding to the cluster as shown in Figure 68. Three different clusters can be distinguished having low Mg-calcite (blue), medium Mg-calcite (red) and high Mg-calcite (yellow).

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7.17 Average spectra according to the cluster analysis performed at level 3 at the anterior edge of the sea urchin tooth of Paracentrotus lividus.

red cluster

blue cluster

yellow cluster

Figure 85: Average spectra corresponding to the cluster as shown in Figure 69. Three different clusters can be distinguished having low Mg-calcite (blue), medium Mg-calcite (red) and high Mg-calcite (yellow).

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7.18 Average spectra according to the cluster analysis performed at the anterior edge of the sea urchin tooth of Paracentrotus lividus in tangential fashion.

red cluster

blue cluster

yellow cluster

Figure 86: Average spectra corresponding to the cluster as shown in Figure 70. Three different clusters can be distinguished having low Mg-calcite (blue), medium Mg-calcite (red) and high Mg-calcite (yellow).

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8 List of Figures

Figure 1: A-C) The three degrees of freedom for a nonlinear molecule (i.e.) water; D-

G) degrees of freedom for a linear molecule (CO2); A, D are symmetrical stretching movements, B, E and F bending or deformation and C, G are asymmetric stretching movements...... 21 Figure 2: The Morse curve illustrating the vibrational levels of an electronic state. The plot shows the typical vibrations having energy on the y-scale and the distance r between the two atoms on the x-scale...... 22 Figure 3: Diagram of possible energy transfer effects. The lowest energy level is at the bottom of the ground electronic state. For IR, absorption of light in a higher level of the ground electronic place takes place. For the Raman effect, much more energy is needed to reach the virtual energy states. For Rayleigh scattering no change in the total energy takes place. For Anti stokes and Stokes scattering either a gain or loss of energy after the scattering process in total takes place. Fluorescence requires even more energy in order to reach the electronic excited state...... 26 Figure 4: Classical Raman spectrum of calcite showing the Rayleigh peak, Stokes and Anti Stokes peaks. As Anti Stokes scattering is less common to Stokes scattering, the peak intensities are weaker...... 27 Figure 5: Scheme of a confocal microscope with a point-like light source. Just the light interacting with the sample in the focal plane is allowed to pass the pinhole, the rest is restricted (reference plane)...... 32 Figure 6: Scheme and image of the WiTec Alpha 300 AR+ system showing the most important parts of the system...... 38 Figure 7: Measurement possibilities offered by the WiTec 300 AR+: A) line scan in xy direction; B) line scan in z direction; C) mapping in xy direction; D mapping in xz direction; E) mapping in yz direction; F) stacked scan (multiple xy map at different z position); G) mapping of a tilted sample via tilt correction method; H) mapping of difficult topography samples via 5x5 surface point correction...... 39 Figure 8: Raman spectral mapping: A) the application of a point array on the area; B) Application of an integral on an interesting peak in each spectrum of the map; C) The resulting intensity color coded Raman false color map...... 42

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Figure 9: The different ways in point distance calculation: A) Euclidean distance determination; B) Manhattan distance calculation...... 45 Figure 10: The reference spectra used for identification: A) calcite of geological calcite single crystal; B) biogenic ACC out of sternal ACC deposits of Porcellio scaber; C) alpha Chitin out of crab shell; D) synthetic hydroxyapatite (HA); E) ACP out of pleoventral phosphate deposits of Tylos europaeus; The colored peaks are used in the case of Raman spectral mapping to represent a distinct peak or component...... 48 Figure 11: The Bravais lattice of calcite and polarization dependent calcite vibrations: A) Calcite Bravais lattice showing the orientation of carbonate and calcium ions. The c- axis is highlighted and the laser polarization directions with respect to shown crystal plane are depicted. B) Two calcite spectra measured along the c-axis at 0° and 90° laser polarization. The calcite librational vibration at 281 cm-1 and the carbonate stretching vibration at 1088 cm-1 are illustrated graphically...... 52 Figure 12: Changes of the integral intensities in calcite due to different laser polarization angles. A) Integral intensity of the calcite librational vibration (black curve) and the carbonate stretching vibration (red curve) depending on the laser polarization angle. B) The CR value for each polarization angle using the values of A) for ratio determination...... 52 Figure 13: The Raman spectra of different carbonate species within a range from 0 to 1150 cm-1. It has to be noted that all spectra were recorded with the 1800 groves/mm grating. All samples are of geological origin: A) calcite (CaCO3, rhombohedral); B) dolomite (Ca,Mg)(CO3)2; C) magnesite (MgCO3) and D) aragonite (CaCO3, orthorhombic). The type of vibrations can be looked up in Table 7...... 55 Figure 14: The tergite cuticle of an isopod showing the pleon and 7 thoracomeres in section A), the surface of a thoracomere showing the anterior and posterior part of a thoracomere (B) and a sagittal cut through a thoracomere in section C, D) the hierarchical arrangement within a tergite cuticle showing several hierarchical levels from the distal to proximal side...... 59 Figure 15: The 7 hierarchical levels forming the chitin protein matrix; A) N-acetyl- glucosamine molecules; B) α-chitin chains; C) chitin-protein nano-fibrils; D) chitin- protein fiber bundles; E) chitin-protein planes; F) twisted plywood like structure; G)

156 optical image showing the twisted plywood like structure within the endocuticle of Helleria brevicornis. [20] ...... 60 Figure 16: Normalized spectra of the 4 important sections within the isopod tergite cuticle. Helleria brevicornis was chosen as an example: The hierarchical levels starting from the most distal regions are epicuticle, exocuticle, endocuticle and membranous layer...... 63 Figure 17: Chitin fiber orientation within the endocuticle and membranous layer of a decalcified tergite cuticle sample of Helleria brevicornis. Two different incident laser light orientations (0° and 90° polarization) were used and the plotted lines were chosen for cross section determination using the corresponding color in Figure 19. The integrated vibration for these two Raman maps is the N-H vibration in the range of 3170-3370 cm-1 using a yellow color code. The cross in each spectrum marks the position of the spectra extracted for comparison in Figure 18...... 66 Figure 18: Normalized average spectra as marked via a cross in Figure 17 at 0° (magenta spectrum) and 90° (red spectrum) incident laser light polarization. A full interpretation can be found in Table 4 (3.2.4)...... 67 Figure 19: Peak intensity distribution of the N-H vibration over the cross sections as marked in Figure 17. Endocuticle and membranous layer are highlighted. The stacking height can be determined via calculation of the distance between two intensity maxima or minima...... 68 Figure 20: Raman maps showing the distribution of organic matter (integration of the C-H stretching vibration from 2800-3100 cm-1) in the exocuticle for 5 different isopod species using a green intensity color code for visualization: A) Helleria brevicornis; B) Porcellio scaber; C) Armadillidium vulgare; D) Tylos europaeus; E) Sphaeroma serratum...... 69 Figure 21: Cross sections over the Raman map of the C-H stretching vibration showing epi- and endo-cuticle as well as membranous layer. The cross sections of all 5 investigated isopod species are shown. The distance between two peak maxima in the endocuticle corresponds to the stacking height of the chitin protein fibers. Increasing distance equals a movement more in the proximal region of the endocuticle. The epicuticle, endocuticle and membranous layer for each species are highlighted using colored boxes...... 71

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Figure 22: The average Raman spectrum of the transitional region between exocuticle and endocuticle within the tergite cuticle of Sphaeroma serratum, showing a mixed spectrum of calcite and chitin...... 72 Figure 23: Raman maps showing the distribution of calcite (integration of the calcite librational vibration) in the exocuticle for 5 different isopod species using a red intensity color code. Quite different kinds of calcite formations can be found: A) Helleria brevicornis; B) Porcellio scaber; C) Armadillidium vulgare; D) Tylos europaeus; E) Sphaeroma serratum. Average spectra of the certain regions can be found in appendix section 7.3...... 74 Figure 24: Sagitally prepared tergite cuticle sample of Porcellio scaber, investigated with polarized confocal Raman mapping using 0° (A-C) and 90° (D-F) incident laser light polarization orientation: A, D) Raman map resulting after integration of the calcite librational vibration; B, E) the carbonate stretching vibration; C, F) map showing the distribution of the calculated CR values using an intensity color code ranging from 0 to 0.8; G) Averaged regions having similar CR values...... 76 Figure 25: Tangential prepared tergite cuticle sample of Porcellio scaber, Raman mapping using 0° (A-C) and 90° (D-F) incident laser polarization orientation: A, D) Raman map due to integration of the calcite librational vibration; B, E) carbonate stretching vibration; C, F) distribution of the calculated CR values using an intensity multi-color code ranging from 0 to 0.8; G) averaged regions with equal CR values. .... 77 Figure 26: Sagitally prepared tergite cuticle sample of Armadillidium vulgare, investigated with polarized confocal Raman mapping using 0° (A-C) and 90° (D-F) incident laser light polarization orientation: A, D) Raman map resulting after integration of the calcite librational vibration; B, E) integration of the carbonate stretching vibration; C, F) map showing the distribution of the calculated CR values using an intensity multi-color code ranging from 0 to 0.8; G) Averaged regions with equal CR values...... 79 Figure 27: Tangential prepared sample of the tergite cuticle of Armadillidium vulgare. The sample was investigated with polarized confocal Raman mapping using 0° (A-C) and 90° (D-F) incident laser light polarization orientation: A, D) Raman map resulting after integration of the calcite librational vibration; B, E) integration of the carbonate stretching vibration; C, F) maps showing the distribution of the calculated CR values

158 using an intensity multi-color code ranging from 0 to 0.5; G) Averaged regions having same CR values are shown. The corresponding CR values are depicted in Table 12. ... 80 Figure 28: The carbonate ratio and averaged CR regions for the anterior and posterior edge of Tylos europaeus at parallel incident laser light orientation. A multi-color color code ranging from 0 to 0.5 was chosen for CR map visualization: A) CR for the anterior thoracomere edge; B) CR for the posterior thoracomere edge; C) averaged CR-regions of the anterior edge; D) averaged CR-regions of the posterior edge. Calculated CR values can be found in appendix 7.4 within Table 15...... 82 Figure 29: Tangential tergite cuticle sample of Tylos europaeus. Samples were investigated with polarized confocal Raman mapping using 0° (A-C) and 90° (D-F) incident laser light polarization: A, D) map after integration of the calcite libr. vibr.; B, E) integration of the carbonate stretching vibr.; C, F) distribution of the calculated CR values ranging from 0 to 0.5; G) Averaged regions having equal CR values...... 83 Figure 30: Sagitally prepared sample of Helleria brevicornis. The sample was investigated with polarized confocal Raman mapping using 0° (A-C) and 90° (D-F) incident laser light polarization angles: A, D) Raman map integrating the calcite librational vibration; B, E) the carbonate stretching vibration; C, F) distribution of the calculated CR values using an intensity multi-color code ranging from 0 to 0.5; G) Averaged regions having equal CR values. Table 16 in section 7.5 is listing all calculated CR values...... 85 Figure 31: Sagitally prepared sample of Sphaeroma serratum: The sample was investigated with polarized confocal Raman mapping using 0° (A-C) and 90° (D-F) incident laser polarization orientation: A, D) Raman map resulting after integration of the calcite librational vibration; B, E) integration of the carbonate stretching vibration; C, F) map showing the distribution of the calculated CR values using an intensity multi- color code ranging from 0 to 0.7; G) Averaged regions having equal CR values...... 86 Figure 32: Raman maps showing the distribution of carbonate (integration of the carbonate stretching vibration) for 5 different isopod species using an orange intensity color code: A) Helleria brevicornis; B) Porcellio scaber; C) Armadillidium vulgare; D) Tylos europaeus; E) Sphaeroma serratum...... 88 Figure 33: Raman maps showing the distribution of phosphate (integration of the phosphate stretching vibration) for 4 different isopod species using a cyan color code:

159

A) Helleria brevicornis; B) Porcellio scaber; C) Armadillidium vulgare; D) Tylos europaeus; Average spectra of the bright parts were calculated and can be looked up in appendix 7.6. For Sphaeroma serratum, it was not possible to detect phosphate...... 89 Figure 34: The anterior (A-E) and posterior (F-J) edge of the tergite cuticle of Tylos europaeus: A, F) microscope image of the anterior and posterior edge, Raman maps are representing several components of the corresponding edge.; B, G) integration of the calcite librational vibration; C, H) integration of the carbonate stretching vibration; D, I) organic (integration of the C-H stretching vibration); E, J) integration of the phosphate stretching vibration...... 91 Figure 35: SEM-images showing an overview of the joint head cuticle of Porcellio scaber; A) location of the joint head; B) the inner and outer side of the joint head, additionally a cross section through broadside of the joint head showing horse shoe like character; C: A cut along the long axis of the joint head. [48] ...... 92 Figure 36: The joint head cuticle of Porcellio scaber as shown in Figure 35B: Each section consists out of 3 Raman maps; Following peaks are integrated showing different components A) integration of the calcite librational vibration; B) integration of the carbonate stretching vibration; C) C-H stretching vibration, D) phosphate stretching vibration; The major structural elements are highlighted and several areas are marked with consecutive numbers and their position (outer edge = o, central region= c, inner edge = i). 1) distal exocuticle, 2) proximal exocuticle, 3) insular calcite regions within the endocuticle at both edges, additionally ACC and chitin protein matrix can be detected, 4) endocuticle, 5) membranous layer, 6) high organic content at both edges, 7) high phosphate content at both edges...... 94 Figure 37: The outer edge of the joint head cuticle. For calcite orientation determination, CR maps at 0° and 90° incident laser light polarization were calculated: A) CR at 0° and B) CR at 90° incident laser light orientation; C) sections having same calcite crystalline orientation according to their CR value. [48] ...... 95 Figure 38: The joint head cuticle of Porcellio scaber prepared at the long side as shown in Figure 35B: A) microscope image of the joint head cuticle showing the connection up to the coxal plate; B) integration of the calcite librational vibration; C) integration of the carbonate stretching vibration; D) C-H stretching vibration showing the distribution of organic matter within the epicuticle, endocuticle and membranous layer; E) phosphate

160 stretching vibration; F) CR-map at parallel laser light orientation using a multi-color code from 0 to 0.5. Important regions are highlighted. 1) insular calcite region within the endocuticle at the edge, 2) distal and proximal exocuticle, 3) wave-like character near the inner edge, 4) membranous layer...... 96 Figure 39: Raman maps showing the joint head cuticle on the broadside (short part) of Helleria brevicornis. Following peaks are integrated showing different components: A) integration of the calcite librational vibration; B) integration of the carbonate stretching vibration; C) organic chitin protein matrix (C-H vibration); D) phosphate stretching vibration; The major structural elements are highlighted and several areas are labelled with consecutive numbers and their position (outer edge = o, central region= c, inner edge = i); 1) exocuticle, 2) endocuticle showing curved arrangement, 3) epicuticle and antenna, 4) organic features at both edges ensuring flexibility, 5) membranous layer surrounding the inner side of the joint head cuticle, 6) high phosphate region at both edges. Average spectra can be found in appendix 7.8...... 98 Figure 40: Raman maps showing the joint head cuticle on the broadside (short part) of Tylos europaeus, Following peaks are integrated showing different components: A) integration of the calcite librational vibration; B) integration of the carbonate stretching vibration; C) organic chitin protein matrix (C-H vibration); D) phosphate stretching vibration; Several interesting regions within the Raman maps are marked and labelled via ongoing numbers; 1) exocuticle, 2) endocuticle showing curved arrangement, 3) epicuticle and antenna, 4) organic features at both edges ensuring flexibility, 5) membranous layer surrounding the inner side of the joint head cuticle, 6) high phosphate region at both edges. Average spectra of the main sections can be found in appendix section 7.9...... 99 Figure 41: The coxal plates of Helleria brevicornis (A-C) and Tylos europaeus (D-F). A, D) integration of the carbonate stretching vibration; B, E) organic chitin protein matrix (C-H vibration); C, F) phosphate stretching vibration; Several interesting regions are marked with consecutive numbers: 1) carbonate enriched region, 2) lower carbonate content, 3) high organic content within the coxal plate, 4) phosphate enriched region, 5) low organic content, 6) high organic content surrounding the coxal plate. Average spectra of certain regions can be found in appendix section 7.10 for Helleria brevicornis and 7.11 for Tylos europaeus...... 101

161

Figure 42: The cornea cuticle of Ligia oceanica (A, B) and Sphaeroma serratum (C, D) showing different number and shapes of the ommatidia. A, C) optical image of the head capsule and the transition into the cornea cuticle having different numbers of ommatidia, abbreviations (hc= head capsule, cc= cornea cuticle); B, D) magnification in the cornea cuticle region where the ommatidia are located...... 102 Figure 43: The transitional region of head capsule of Ligia oceanica to the ommatidial array having several components according to the illustrated Raman maps; A) map after integrating the calcite librational vibration; B) integration of the carbonate stretching vibration; C) organic material (C-H stretching vibration); D) carotenoids (integration of C=C vibration); Several interesting regions are marked with ongoing numbers: 1) exocuticle within the head capsule, 2) exocuticle at the transition from head capsule to cornea cuticle, 3) exocuticle at an ommatidium, 4) endocuticle, 5) membranous layer, 6) carotenoids within the endocuticle of the head capsule...... 103 Figure 44: The average Raman spectra of the exocuticle (Figure 43A 1, 2), endocuticle (Figure 43B, C 4), the membranous layer (Figure 43C 5) and the carotenoid enriched area (Figure 43D 6) in the center of the head capsule of Ligia oceanica...... 104 Figure 45: Raman maps showing several components of 4 ommatidia segments of Ligia oceanica. A) integration of the calcite librational vibration; B) integration of the carbonate stretching vibration; C) organic material (C-H vibration); D) phosphate stretching vibration; E) Cluster analysis of a Raman map of 2 ommatidia allowing an even further discrimination in the content of the present components. Average spectra of the various regions of the cluster analysis can be found in Figure 46...... 105 Figure 46: The average spectra resulting after performing a Cluster analysis on the Raman map of the cornea cuticle of Ligia oceanica as shown in Figure 45E...... 106 Figure 47: The transitional region of head capsule of Sphaeroma serratum towards the ommatidial array having several components according to the illustrated integrated Raman maps.: A) integration of the calcite librational vibration; B) carbonate stretching vibration; C) organic material (C-H stretching vibration); D) carotenoid (C=C vibration); Several interesting regions are marked with consecutive numbers: 1) exocuticle within the head capsule, 2) exocuticle at the transition from head capsule to cornea cuticle, 3) exocuticle at an ommatidium, 4) endocuticle, 5) membranous layer, 6) carotenoids within the endocuticle of the head capsule...... 107

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Figure 48: Raman maps showing several components of 11 ommatidial segments of Sphaeroma serratum (A-C). D-G are showing a single ommatidium for a more detailed analysis of the structure and G is representing a cluster analysis. A, D) integration of the calcite librational vibration representing calcite; B, E) integration of the carbonate vibration representing ACC and calcite; C, F) integration of the C-H stretching vibration representing organic material; G) Cluster analysis of the present Raman map allowing an even further discrimination in the content of the present components; The corresponding spectra are shown in appendix section 7.13...... 109 Figure 49: The masticator apparatus of the sea urchin tooth of Paracentrotus lividus: A) view from above the masticator showing 5 T-shaped sea urchin teeth; B) view at the tips of the 5 teeth; C) view from aside the masticator apparatus; D) one tooth element showing the how the tooth is fixed by the help of the jaw bone on the outside and the inside of the masticator apparatus; The jaw bone functions as an encasement and guiding tool for the tooth. The lower part of the tooth is named keel...... 111 Figure 50: General scheme of the sea urchin tooth: A) Cross section in the central region showing 5 different structural elements. primary plates (pp), secondary plates (sp) stone, carinar process plates (cpp) and prisms (pr); B) cross section near the anterior and the posterior edge of the tooth showing different maturation stages of the tooth...... 112 Figure 51: Overview of on the masticator apparatus from above showing the 5 convoluted teeth reaching the plumula. The central region of an intact tooth is magnified to demonstrate the sample preparation in order to investigate the start of the tooth formation...... 113 Figure 52: Cross section of the sea urchin tooth of Paracentrotus lividus at the posterior end (plumula) showing 4 primary plates in their early formation stage. A) microscope image showing the cross section; B) Raman map after integration of the calcite librational vibration; C) Raman map after integration of the carbonate stretching vibration; D) the peak position distribution of the carbonate stretching vibration and the three areas used for average spectrum acquisition, which are shown in Figure 53; E) the distribution of the FWHM of the carbonate stretching vibration and schematically the growth directions of the primary plates...... 114

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Figure 53: The average spectra corresponding to the areas marked in Figure 52D. The red spectrum is showing a high calcite content, whereas the blue and the green spectrum are showing significant ACC content. For conformation, additionally an orange ACC spectrum is shown and its contribution to the high baseline feature to the spectra highlighted via the hatched area from 90-300 cm-1...... 115 Figure 54: Cluster analysis of a Raman map with corresponding average spectra 200 µm deeper compared to section 1 as described in Figure 52. The calcite librational vibration and the carbonate stretching vibration were chosen as basis for the cluster analysis...... 117 Figure 55: The distribution of calcium carbonate material within the third polished region starting from the plumula. A) integration of the calcite librational vibration; B) integration of the carbonate stretching vibration; C) the calculated peak center of the carbonate stretching vibration (1080-1092 cm-1), three interesting areas are marked showing the corresponding normalized average spectra in Figure 56; D) The calculated FWHM of the carbonate stretching vibration (18-25 cm-1)...... 118 Figure 56: The average spectra from several areas within section 3 of the polished sea urchin tooth. The spectra are color coded analogue to the areas as shown in Figure 55C. Red= spectrum in the central region of the primary plates, Blue= at the edge of the primary plates (growth direction of further primary plates), Green= beginning of the secondary plate formation...... 119 Figure 57: The distribution of calcite crystalline material in the formation of the sea urchin tooth of Paracentrotus lividus. Level 4 is showing the consolidation of the so far separated primary plate units. A) integration of the calcite librational vibration; B) integration of the carbonate stretching vibration; C) center of the carbonate stretching vibration determined via Gauss fit (1080-1092 cm-1) Regions used for average spectrum determination are highlighted in their corresponding color, red= representive for fully crystallized Mg-calcitic material, blue= formation of the secondary plates showing detectable ACC content; D) cluster analysis showing fully crystallized (red) and partly crystallized calcium carbonate...... 120 Figure 58: Two important average spectra according to the areas marked in Figure 57C, showing fully crystallized material (red spectrum) and the formation of the secondary plates (blue spectrum), indicating the presence of ACC...... 121

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Figure 59: The sea urchin tooth of Paracentrotus lividus. Raman maps showing level 5 in depth with respect to the posterior edge in the plumula. Continuation of the growth and formation of the secondary plates as well as start of the stone formation in the center of the tooth are illustrated: A) Raman map showing the integral intensity of the calcite librational vibration; B) the carbonate stretching vibration; C) The center of the carbonate stretching vibration (1080-1095 cm-1); D) Cluster analysis showing different calcite maturation levels...... 122 Figure 60: Average spectra corresponding to the cluster as shown in Figure 59. Three different clusters can be distinguished having fully crystallized calcite (red), partially crystallized calcite (cyan) and minimal crystallized calcite, but more ACC (yellow). 123 Figure 61: The sea urchin tooth of Paracentrotus lividus. Raman maps showing level 6 in depth with respect to the posterior edge in the plumula. Continuation of the growth and formation of the secondary plates and the stone are illustrated: A) Raman map showing the integral intensity of the calcite librational vibration; B) the carbonate stretching vibration; C) The center of the carbonate stretching vibration (1080-1095 cm- 1); D) Cluster analysis showing different calcite maturation levels (spectra in Figure 62)...... 124 Figure 62: Average spectra corresponding to the cluster as shown in Figure 61. Three different clusters can be distinguished having fully crystallized calcite (red), partially crystallized calcite (cyan) and minimal crystallized calcite, but more ACC (yellow). 125 Figure 63: The central region of the sea urchin tooth: A) microscope image of central region of the tooth. Marked areas are the regions chosen for Raman mapping; B) Integration of the carbonate stretching vibration of section 1 showing the increasing size of the calcite fibers converting in the so named prisms...... 126 Figure 64: The central region of the sea urchin tooth of Paracentrotus lividus. Raman maps of section 2 and 3 according to Figure 63. A-C correspond to section 2, D, E correspond to section 3: A, D) integration of the calcite librational vibration; B, E) integration of the carbonate stretching vibration; C) distribution of the peak center of the carbonate stretching vibration determined via Gauss fits (1080-1095 cm-1); Average spectra of stone, prisms and carinar process plates of section 2 as well as prims and carinar process plates of section 3 are shown in appendix section 7.15...... 127

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Figure 65: Microscopic images of the anterior edge of the sea urchin tooth of Paracentrotus lividus: A) view on a tangential prepared tooth showing the progression in abrasion of the keel towards the tip as well as the primary plates and the stone; B) view on a polished tooth sample from in front of the tooth after just polishing 20 µm from the final end of the tooth revealing just stone material and a small amount of primary plates...... 128 Figure 66: The anterior edge of the sea urchin tooth of Paracentrotus lividus. Polishing level 1 was at 20 µm distance from the ultimate end of the tooth: A) integration of the calcite librational vibration; B) integration of the carbonate stretching vibration; C) center of the carbonate stretching vibration calculated via Gauss fits (1080-1095 cm-1) and D) cluster analysis revealing three different clusters...... 129 Figure 67: Average spectra corresponding to the cluster as shown in Figure 66. Three different clusters can be distinguished having low Mg-calcite (blue), medium Mg- calcite (red) and high Mg-calcite (yellow)...... 130 Figure 68: The anterior edge of the sea urchin tooth of Paracentrotus lividus. Polishing level 2 was 200 µm deeper into the tooth material with respect to level 1: A) integration of the calcite librational vibration; B) integration of the carbonate stretching vibration; C) center of the carbonate stretching vibration calculated via Gauss fits (1080-1095 cm- 1) and D) cluster analysis revealing three different clusters...... 132 Figure 69: The anterior edge of the sea urchin tooth of Paracentrotus lividus. Polishing level 3 was 200 µm deeper into the tooth material with respect to level 2: A) integration of the calcite librational vibration; B) integration of the carbonate stretching vibration; C) center of the carbonate stretching vibration calculated via Gauss fits (1080-1095 cm- 1) and D) cluster analysis revealing three different clusters. The corresponding spectra can be found in appendix section 7.17...... 133 Figure 70: The sea urchin tooth of Paracentrotus lividus. Tangentially prepared sample showing structural elements with different Mg2+ content in the calcite crystalline lattice after Raman spectral mapping. A) integration of the calcite librational vibration; B) carbonate stretching vibration; C) peak center of the carbonate stretching vibration determined via Gauss fits (1080-1095 cm-1); D) cluster analysis showing high (yellow), medium (red) and low (blue) Mg-calcite...... 134

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Figure 71: Normalized average spectra of the endocuticle of different investigated isopod species like Helleria brevicornis, Porcellio scaber, Armadillidium vulgare, Tylos europaeus and Sphaeroma serratum as mentioned in the results and discussion part. ACC, chitin protein fibers and phosphate are dominating the average spectra determined in this region...... 137 Figure 72: Normalized average spectra of the membranous layer of different investigated isopod species like Helleria brevicornis, Porcellio scaber, Armadillidium vulgare, Tylos europaeus and Sphaeroma serratum as mentioned in 4.3.2.2...... 138 Figure 73: Normalized average spectra of the exocuticle of different investigated isopod species like Helleria brevicornis, Porcellio scaber, Armadillidium vulgare, Tylos europaeus and Sphaeroma serratum as mentioned in 4.3.3.1...... 139 Figure 74: Normalized average spectra of the phosphate enriched parts of different investigated isopod species like Helleria brevicornis, Porcellio scaber, Armadillidium vulgare and Tylos europaeus as mentioned in the results and discussion part...... 142 Figure 75: The joint head cuticle of Porcellio scaber: Spectra from average regions a-e are shown at laser polarization of 90°, Furthermore, the high phosphate region in the outer edge as well as the membranous layer...... 143 Figure 76: The joint head cuticle of Helleria brevicornis: Average spectra of classical elements are shown, which are exo-, endo-cuticle as well as membranous layer...... 144 Figure 77: The joint head cuticle of Tylos europaeus: Average spectra of classical elements are shown, which are exo-, endo-cuticle as well as membranous layer...... 145 Figure 78: The coxal plates of Helleria brevicornis: Calculated average spectra of the carbonate enriched region 1 (red spectrum) and the lower part of the coxal plate having lower carbonate content (blue spectrum = region 2) ...... 146 Figure 79: The average spectra of three selected region within the coxal plates of Tylos europaeus: A) Average over the whole carbonate map as depicted in Figure 41D, B) Average spectrum of the small central region having low organic content in comparison to the rest of the coxal plate (Figure 41E,5), C) Average spectrum of the very small region having higher phosphate content (Figure 41F,4)...... 147 Figure 80: The average Raman spectra of the exocuticle, endocuticle, the organic protein matrix and the carotenoid enriched area in the center of the head capsule of Sphaeroma serratum...... 148

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Figure 81: The average spectra resulting after performing a Cluster analysis of the cornea cuticle of Sphaeroma serratum as shown in Figure 48G...... 149 Figure 82: The average spectra of the central (black spectrum) and edge (red spectrum) at level 1 within the posterior edge of the sea urchin tooth of Paracentrotus lividus. . 150 Figure 83: Average spectra of the central region of certain areas like stone, prisms as well as carinar process plates. The spectra are originating from different sections as already depicted in Figure 63 and Figure 64...... 151 Figure 84: Average spectra corresponding to the cluster as shown in Figure 68. Three different clusters can be distinguished having low Mg-calcite (blue), medium Mg- calcite (red) and high Mg-calcite (yellow)...... 152 Figure 85: Average spectra corresponding to the cluster as shown in Figure 69. Three different clusters can be distinguished having low Mg-calcite (blue), medium Mg- calcite (red) and high Mg-calcite (yellow)...... 153 Figure 86: Average spectra corresponding to the cluster as shown in Figure 70. Three different clusters can be distinguished having low Mg-calcite (blue), medium Mg- calcite (red) and high Mg-calcite (yellow)...... 154

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9 List of tables

Table 1: Available media and their correspondent refractive index nd. [28] ...... 31 Table 2: Available microscope objectives with different magnification and their numerical aperture, working distance and the calculated lateral (rmin) and axial (zmin) resolution determined according to equation (12) and (14)...... 31 Table 3: The connection between spatial resolution, the number of spectra and the resulting time in case of a 50x50 µm Raman spectral map at an integration time of 1 second...... 41 Table 4: Peak characterization of the reference spectra as shown in Figure 10. Literature references are listed...... 49 Table 5: Peak integration ranges and the applied color code used for Raman spectral mapping in order to illustrate the distribution of several components...... 50

Table 6: Determination of the MgCO3 content in Mg-calcite due to the peak position of the carbonate stretching vibration of different isopod species. A comparison with XRD results is shown...... 54 Table 7: Fully interpretation of the Raman spectra as shown in Figure 13...... 55 Table 8: Investigated species with their characteristically habitat, the type of defense mechanism and the thickness of the exocuticle...... 58 Table 9: total chemical composition of the tergite cuticle of the 5 investigated isopod species...... 61 Table 10: The thickness of different regions within the tergite cuticle of all 5 investigated species. *diameter was determined with underlying samples of Figure 20...... 70 Table 11: Overview of the resulting carbonate ratio values estimating the orientation of the regions as depicted in Figure 24G and Figure 25G for Porcellio scaber in sagittal and tangential perspective...... 78 Table 12: Overview of the resulting carbonate ratio values estimating the orientation of the regions as depicted in Figure 26 and Figure 27 for Armadillidium vulgare in sagittal and tangential prepared samples...... 81 Table 13: Overview of the resulting carbonate ratio values estimating the orientation of the regions as depicted in Figure 31...... 86

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Table 14: CR-values for a sagitally polished joint head cuticle sample of Porcellio scaber. Polarized Raman mapping was performed at 0° and 90° incident laser light polarization. The CR-values at each polarization and area were determined...... 95 Table 15: CR values for Tylos europaeus edges in sagittal fashion at parallel polarization and a tangential polished sample at parallel and perpendicular incident laser light polarization...... 139 Table 16: CR-values for a sagittal polished exocuticle sample of Helleria brevicornis. Polarized Raman mapping was performed at 0° and 90° incident laser light polarization. The CR-values at each polarization and area were determined...... 141

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