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Morphology and Dynamics of Ice Crystals and the Effect of Proteins ! ! University of the Basque Country !"#$%&'"(&)*+),$&"%-$.)/012-32) PhD thesis Morphology and dynamics of ice crystals and the effect of proteins Presented by María Cascajo Castresana Supervised by Prof. Alexander M. Bittner & Prof. Silvina Cerdeny ! Donostia/San Sebastián, 2017 (cc)2017 MARIA CASCAJO CASTRESANA (cc by-nc 4.0) This PhD thesis has been performed at, "#$%!Ȃ&''#()$*!+,-./! 010!232-+456!! 7-2-'893:"32!"#)3'89;2! Polymers and Soft Matter Group Materials Physics Center (CFM) Donostia/San Sebastián ! ! Institute for Atmospheric and Climate Science ETH Zürich ! ! ! ! ! ! ! ! ! !!!!!!! ! !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! ! !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! ! !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!To the memory of my grandparents, Ricardo and Amelia Contents ! Summary ...................................................................................... 5 Resumen ...................................................................................... 9 Chapter 1: Introduction««««««««««««««5 1. Motivation and objectives ............................................................................. 16 1.1. Basic concepts ................................................................................. 19 1.1.1. Phase diagrams ............................................................... 19 1.1.2. Vapour pressure.............................................................. 20 1.1.3. Humidity .......................................................................... 23 1.1.4. Quasi-liquid layers .......................................................... 24 1.1.5. Diffusional dynamics and its analysis ........................... 30 1.2. Properties of ice ............................................................................... 34 1.2.1. Hexagonal ice Ih .............................................................. 37 1.2.2. Ice crystals ....................................................................... 38 1.2.3. The ice-vapour interface ................................................ 43 1.3. Ice nucleation in the atmosphere ..................................................... 45 1.3.1. Homogeneous ice nucleation .......................................... 45 1.3.2. Heterogeneous ice nucleation and its mechanisms ...... 46 1.3.3. Biological aerosols and their role in ice nucleation ..... 48 1.4. The geo- and astrophysical relevance of ice ................................... 53 ! "! ! Contents 1.4.1. Excursus: Ice on Mars .................................................... 54 1.4.2. Excursus: Ice on three moons ........................................ 56 1.5. References .................................................................................................... 59 Chapter 2: Electron microscopy of ice deposition and sublimation«««««««««««««««««««71 2.1. The Environmental Scanning Electron Microscope (ESEM) ....... 72 2.2. ESEM imaging .................................................................................. 76 2.2.1. Contrast ........................................................................... 80 2.2.2. Beam damage .................................................................. 82 2.3. Growth and sublimation processes ................................................. 85 2.3.1. Single crystals and merging ........................................... 85 2.3.2. Polycrystalline ice and grain boundaries ...................... 89 2.3.3. Sublimation ...................................................................... 92 2.4. Unusual microscale features observed during sublimation .......... 99 2.5. Conclusions and future prospects ................................................... 102 2.6. References .......................................................................................... 105 Chapter 3: Imaging and particle tracking analysis of the quasi-liquid layer««««««««««««««««1 3.1. Experimental part .............................................................................. 112 3.2. Preliminary ESEM tests for ice surface dynamics at high T (close to 0ºC) ....................................................................................... 116 3.3. Nanoparticle tracking method .......................................................... 118 #! ! 3.3.1. Tracking analysis ............................................................ 123 3.3.2. MATLAB code ................................................................ 125 3.4. Nanoparticle tracking results ........................................................... 127 3.4.1. Movement type I ............................................................. 129 3.4.2. Movement type II ............................................................ 132 3.5. Conclusions and future prospects .................................................... 137 3.6. References ........................................................................................... 140 Chapter 4: Ice nucleation in presence of proteins««««3 4.1. Proteins and virus description ......................................................... 144 4.2. Experimental techniques .................................................................. 151 4.2.1. Drop freezing technique ................................................. 151 4.2.2. Differential scanning calorimetry (DSC) ...................... 155 4.3. Ice nucleation of proteins ................................................................. 157 4.3.1. Sample preparation ........................................................ 157 4.3.2. Results .............................................................................. 158 4.4. Ice nucleation of apoferritin and ferritin ....................................... 162 4.4.1. Sample preparation ........................................................ 162 4.4.2. Results in water ............................................................... 163 4.4.3. pH variations ................................................................... 171 4.4.4. Heating experiments ....................................................... 186 4.5. DSC measurements ............................................................................ 196 4.5.1. DSC results on pure water .............................................. 196 ! $! ! Contents 4.5.2. DSC results on protein solutions .................................... 200 4.6. Conclusions and future prospects .................................................... 206 4.7. References ........................................................................................... 211 Appendices««««««««««««««««««««««««««« I. Signal detection in the ESEM ................................................................................... 217 II. Optimization ESEM setup ...................................................................................... 221 III. Software tools for video processing ...................................................................... 225 IV. Random walk tests .................................................................................................. 239 V. List of analysed videos with nanoparticle movement detection ........................... 243 VI. MATLAB codes for drop freezing images processing ........................................ 259 VII. Frozen fraction plots of tested proteins and virus ............................................. 269 VIII. Apoferritin and ferritin sample solutions appearance ..................................... 275 Acronyms & Abbreviations ......................................................................................... 281 Acknowledgments ......................................................................................................... 285 %! ! Summary The thesis "Morphology and dynamics of ice crystals and the effect of proteins" is based on a wide range of themes, from the basics of ice structure (such as ice surface and ice morphology), over the interaction of proteins with ice, to environmental science (which encompasses cloud formation and glacier dynamics). The focus is on interfaces, namely ice/vapour and ice/water. By extending environmental scanning electron microscopy (ESEM) to unusually low temperatures, areas in the pressure-temperature landscape of ice morphologies were accessed in-situ. This means recording real time ESEM images and movies under full humidity control in a dynamic ice-vapour equilibrium. Besides reproducing known morphologies of single crystalline and of polycrystalline ice, the high time resolution (frame rate 1 Hz) gives access to dynamic growth and sublimation events. During these processes, well-known forms of ice crystallites and polycrystalline ice were found. The time-resolved growth of ice is generally rather difficult to control, especially on the microscale. However, difficulties with the method for studying the growth of ice crystals, such as the effect of the electron beam-gas ionization and charging effects, the problem of facilitating repeated and localized ice growth are discussed and successfully overcome. Sublimation events at small undersaturation of water vapour are better accessible. The sublimation rates fit to natural phenomena on ice fields, e.g. in the Antarctica or on Mars. The dynamically evolving shapes are complex: From hexagonal single crystal columns, ! "! ! Summary but also from smooth polycrystalline surfaces, sharp sub-microscale ridges develop, and finally dominate the overall structure. A new geometry are the so-called
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