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File for the Full Video The Structural and Compositional Basis for the Transparency and Physical Properties of the Skogsbergia lerneri Carapace A thesis submitted to Cardiff University for the degree of Doctor of Philosophy March 2020 Benjamin Martin Rumney Structural Biophysics Group School of Optometry and Vision Science Cardiff University Supervisors: Professor Keith Meek Dr Julie Albon Dr Philip Lewis Ben Rumney Acknowledgements and Contents Acknowledgements I would like to firstly thank all of my supervisors for helping me through a thesis with such a unique subject compared to the norm. I would like to thank Professor Keith Meek for supporting me beyond the required level both in and out of the PhD. I would like to thank Dr Julie Albon for being a constant source of help and support throughout the three and half years of the project. I would like to thank Dr Phil Lewis for his help in all things EM-based and his advice through seemingly endless microscope issues. I would like to thank all my friends for their support and for suffering with me effectively going radio silent for the final year of my PhD, as well as all the new friends I have made at Cardiff. All the chats and socials we had amongst both the postgrads and the biophysics group as a whole were a constant and welcome support. I especially want to thank my parents, Martin and Louise Rumney and my sister Abi, who have supported me both emotionally and practically throughout the entire project as they have throughout all my life. I would also like to thank Dr Siân Morgan for all her assistance throughout the ostracod project as well as all our trips to and from Diamond, as well as everyone in the Biophysics team for always being friendly and happy to help with anything. I would also like to thank Nick White for his enormous help and advice with all things non-linear.I would like to thank Professor Andrew Parker for his help on both the project as a whole, his specific advice on ostracods and his assistance with locating the ostracod populations that were used in this thesis. I would like to thank both Professor Andrew Parker and Dr Tegwen Malik for their assistance in capturing and transporting the ostracods to Cardiff from Florida. I’m especially grateful after hearing about some of the horror stories they came back with! I would also like to thank Tegwen for all her help with maintaining the ostracod tanks, without which the ostracods would not have thrived as well as they did. Finally I would like to thank DSTL for their funding of this project, allowing me to be able to carry out this PhD in the first place and giving me the opportunity to be able to present my work at numerous speciailsed conferences. ii Ben Rumney Acknowledgements and Contents Table of Contents Acknowledgements ...................................................................................................... ii Table of Contents ........................................................................................................ iii Table of Figures……………………………………………………………………………………………..………vii Abbreviation List………………………………………………………………………………………………….…xi Abstract ..................................................................................................................... xiii 1. Introduction ........................................................................................................ 15 1.1 Skogsbergia lerneri ..................................................................................... 15 1.1.1 Habitats ................................................................................................ 15 1.1.2 Physical Features .................................................................................. 17 1.1.3 Life Cycle................................................................................................. 19 1.2 General Crustacean Integument Architecture and its relationship to Ostracods ................................................................................................................ 21 1.2.1 Composition ......................................................................................... 21 1.2.2 Architecture ......................................................................................... 25 1.3 Physical Properties of Crustacean Carapaces ............................................. 31 1.3.1 Hierarchical Design ............................................................................... 32 1.3.2 Heat Resistance .................................................................................... 35 1.3.3 Hardness ............................................................................................... 36 1.3.4 Hydration.............................................................................................. 37 1.3.5 Elastic Modulus .................................................................................... 38 1.3.6 Fracture Toughness .............................................................................. 39 1.3.7 Transparency ........................................................................................ 40 1.4 Aims and Hypothesis ................................................................................... 43 1.4.1 Overall Hypothesis ............................................................................... 43 1.4.2 Overall Aim ........................................................................................... 43 1.4.3 Hypothesis 1 ......................................................................................... 43 1.4.4 Aim 1 .................................................................................................... 43 1.4.5 Hypothesis 2 ......................................................................................... 44 1.4.6 Aim 2 .................................................................................................... 44 1.4.7 Hypothesis 3 ......................................................................................... 44 1.4.8 Aim 3 .................................................................................................... 44 1.4.9 Hypothesis 4 ......................................................................................... 44 iii Ben Rumney Acknowledgements and Contents 1.4.10 Aim 4 .................................................................................................... 45 2. Methodology ...................................................................................................... 47 2.1 Aquaculture ................................................................................................. 47 2.1.1 Licensing for the Acquiring and Transportation of Samples................ 47 2.1.2 Transfer of Ostracods from Deliveries to Aquaculture Tanks ............. 47 2.1.3 Maintenance of Aquaculture ............................................................... 48 2.2 Dissection .................................................................................................... 49 2.2.1 Development of dissection protocol ................................................... 49 2.2.2 Dissection Axes .......................................................................................... 50 2.3 Electron Microscopy .................................................................................... 51 2.3.1 Transmission Electron Microscopy (TEM) ........................................... 51 2.3.2 Serial Block Face Scanning Electron Microscopy ................................. 53 2.3.3 Amira Software .................................................................................... 55 2.3.4 Scanning Electron Microscopy ............................................................. 55 2.4 Non-linear microscopy Sample Preparation ............................................... 56 2.4.1 Cryosectioning ..................................................................................... 56 2.4.2 Butyl:methyl methacrylate (BMMA) .................................................... 57 2.4.3 Equipment and software ..................................................................... 59 2.5 X-ray Microtomography and Ptychography ................................................ 60 2.5.1 X-ray Microtomography ....................................................................... 60 2.5.2 Ptychography ....................................................................................... 61 2.6 Elemental Analysis....................................................................................... 63 2.6.1 Energy-dispersive X-ray spectroscopy ................................................. 63 2.6.2 X-ray Fluorescence ............................................................................... 64 2.6.3 X-ray Absorption Near Edge Structure ................................................ 65 2.6.4 Wide Angle X-ray Scattering (WAXS) ................................................... 66 2.7 Summary of Structural and Compositional Techniques ............................. 67 2.8 Spectroscopy ............................................................................................... 69 2.8.1 Refractometry ...................................................................................... 69 2.8.2 Transmittance measurements ............................................................. 69 3. Ultrastructure analysis........................................................................................ 71 3.1 Introduction................................................................................................
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