FACULTE DE BIOLOGIE ET DE MEDECINE Cryo- electron microscopy of vitreous sections of native biological cells and tissues: Methodology and applications Thèse de doctorat ès sciences de la vie (PhD) présentée à la Faculté de Biologie et de Médecine de l’Université de Lausanne par Ashraf S. Al-Amoudi Physicien diplômé, de l’Université de Birzeit, Palestine Jury Prof. Pierre.Goloubinoff, Président Prof. Jacques Dubochet, Directeur de thèse Dr. Daniel Studer, Expert Dr. Karl Zierold, Expert Lausanne 2004 TABLE OF CONTENTS TABLE OF CONTENTS.......................................................................................................I List of abbreviations............................................................................................................IV Acknowledgements................................................................................................................V Résumé ..................................................................................................................................VI Abstract ...............................................................................................................................V II 1. Introduction ....................................................................................................................... 1 1.1 Water in cell biology ......................................................................................................1 1.1.1 Ordinary bulk water ................................................................................................1 1.1.2 Cell water ................................................................................................................2 1.2 Biological electron microscopy......................................................................................2 1.2.1 Chemical fixation ....................................................................................................3 1.2.2 Cryofixation ............................................................................................................3 1.3 Standard cryotechniques ................................................................................................5 1.4 Cryo-electron microscopy of vitreous sections (CEMOVIS) ........................................7 1.5 Subject of the present thesis...........................................................................................8 2. General methods................................................................................................................ 9 2.1 High- pressure freezing ..................................................................................................9 2.2 Cryo-ultramicrotomy....................................................................................................10 2.3 Cryo-electron microscopy ............................................................................................12 3. Cutting process ................................................................................................................ 13 3.1 Cutting artefacts in vitreous sections ...........................................................................13 3.1.1 Introduction ...........................................................................................................13 3.1.2 Material and methods ............................................................................................14 3.1.3 Results ...................................................................................................................14 3.1.3.1 General aspect of vitreous sections ................................................................15 3.1.3.2 Cut-Rotate-Cut experiments...........................................................................15 3.1.3.3 Cutting direction.............................................................................................16 3.1.3.4 Cutting feed ....................................................................................................17 3.1.3.5 Cutting speed..................................................................................................17 3.1.3.6 Cutting temperature........................................................................................17 3.1.3.7 Block size .......................................................................................................18 3.1.4 Discussion .............................................................................................................18 3.1.4.1 Cutting vitrified samples ................................................................................18 3.1.4.2 Knife marks ....................................................................................................19 3.1.4.3 Compression...................................................................................................20 3.1.4.4 Crevasses........................................................................................................22 3.1.4.5 Chatter ............................................................................................................22 3.1.4.6 Practical optimal cutting conditions...............................................................23 3.1.5 Conclusion.............................................................................................................24 3.2 Cutting- induced amorphous solid water (CIAS).........................................................26 3.3 Oscillating cryo-knife...................................................................................................29 3.3.1 Introduction ...........................................................................................................29 3.3.2 Construction of oscillating cryo-knife...................................................................29 3.3.3 Results and discussion...........................................................................................30 4. Selected biological applications...................................................................................... 32 4.1 Human epidermis .........................................................................................................32 4.1.1 Introduction ...........................................................................................................32 4.1.2 Materials and methods ..........................................................................................33 4.1.2.1 Cryopreparation..............................................................................................33 4.1.2.2 Conventional chemical fixation preparation ..................................................33 4.1.3 Results and discussion...........................................................................................33 4.1.3.1 The general aspect of human epidermis.........................................................33 4.1.3.2 Desmosomes................................................................................................34 4.1.3.3 Keratin intermediate filaments .......................................................................35 4.1.4 Conclusion.............................................................................................................36 4.2 Gram-negative bacteria: Pseudomonas aeruginosa (PAO1)........................................37 4.2.1 Introduction ...........................................................................................................37 4.2.2 Materials and methods ..........................................................................................37 4.2.3 Results and discussion...........................................................................................38 4.2.3.1 The general aspect of Gram-negative bacteria...............................................38 4.2.3.2 Outer and plasma membranes ........................................................................38 4.2.3.3 Peptidoglycan .................................................................................................39 4.2.3.3 Nucleoid .........................................................................................................39 4.2.4 Conclusion.............................................................................................................40 4.3 Cyanobacteria: Lyngbya majuscula .............................................................................41 4.3.1 Introduction ...........................................................................................................41 II 4.3.2 Materials and methods ..........................................................................................41 4.3.3 Results and discussion...........................................................................................41 4.3.3.1 General view of the cynaobacterium Lyngbya majuscula .............................41 4.3.3.2 Cell envelope..................................................................................................42 4.3.3.3 Extracellular matrix........................................................................................43 4.3.4 Conclusion.............................................................................................................43 General conclusion and outlook......................................................................................... 44 Illustrations.......................................................................................................................... 45 References