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Polarized Confocal Raman Microscopy As a Powerful Tool in the Investigation of Biomineralized Systems

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Polarized Confocal Raman Microscopy As a Powerful Tool in the Investigation of Biomineralized Systems 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 2 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 3 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. 4 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 5 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! 6 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 7 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 ........................
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