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Brno University of Technology Vysoké Učení Technické V Brně BRNO UNIVERSITY OF TECHNOLOGY VYSOKÉ UČENÍ TECHNICKÉ V BRNĚ FACULTY OF MECHANICAL ENGINEERING FAKULTA STROJNÍHO INŽENÝRSTVÍ INSTITUTE OF PHYSICAL ENGINEERING ÚSTAV FYZIKÁLNÍHO INŽENÝRSTVÍ SCANNING ELECTRON MICROSCOPY AND ITS APPLICATIONS FOR SENSITIVE SAMPLES RASTROVACÍ ELEKTRONOVÁ MIKROSKOPIE A JEJÍ APLIKACE PRO SENSITIVNÍ VZORKY DOCTORAL THESIS DIZERTAČNÍ PRÁCE AUTHOR Mgr. Kamila Hrubanová AUTOR PRÁCE SUPERVISOR Ing. Vladislav Krzyžánek, Ph.D. ŠKOLITEL BRNO 2019 This doctoral thesis was created at the Institute of Scientific Instruments of the CAS, v. v. i., Electron Microscopy department, under supervision of Ing. Vladislav Krzyžánek, Ph.D. Statement I declare that I have written the Doctoral Thesis titled “Scanning electron microscopy and its applications for sensitive samples” independently, under the guidance of the supervisor Ing. Vladislav Krzyžánek, Ph.D., and using exclusively the technical references and other sources of information cited in the thesis and listed in the comprehensive bibliography at the end of the thesis. As the author I furthermore declare that, with respect to the creation of this Doctoral Thesis, I have not infringed any copyright or violated anyone’s personal and/or ownership rights. In this context, I am fully aware of the consequences of breaking Regulation§11 of the Copyright Act No. 121/2000 Coll. of the Czech Republic, as amended, and of any breach of rights related to intellectual property or introduced within amendments to relevant Acts such as the Intellectual Property Act or the Criminal Code, Act No. 40/2009 Coll., Section 2, Head VI, Part 4. In Brno Kamila Hrubanová Acknowledgements Herein, I would like to express my sincere gratitude to my supervisor Ing. Vladislav Krzyžánek, Ph.D. for his support, motivation and valuable advice during my Ph.D. studies and to all of my colleagues, especially to Mgr. Bc. Kateřina Adámková and Mgr. Ing. Radim Skoupý, because good team is essential in scientific research. I would also like to thank Mgr. Ota Samek, Ph.D. for his advice and support with the Raman spectroscopy results. I would like to thank the staff at the Biological Centre of the CAS in České Budějovice for their support in my initial efforts with the cryo-SEM technique. A very special gratitude goes to my children for their generous patience with my continuous work commitment and to my husband for his endless support, help and understanding in difficult times. Finally, I would like to thank my family and my friends for being in my life and making me the person that I am. Funding: The research was supported by the Czech Science Foundation (projects 19-20697S; 17-15451S; 16-12477S; 15-20645S; 14-20012S; 19-29651L), the Ministry of Industry and Trade of the Czech Republic (project TRIO FV30271), the Technology Agency of the Czech Republic (projects TN01000008; TE01020118) and the Ministry of Education, Youth and Sports of the Czech Republic (projects LO1212; CZ.1.07/2.3.00/20.0103). The research infrastructure was funded by Ministry of Education, Youth and Sports of the Czech Republic and European Commission (project CZ.1.05/2.1.00/01.0017) and by the Czech Academy of Sciences (project RVO:68081731). Kamila Hrubanova is grateful to a ThermoFisher/CSMS scholarship for the support. Abstract The presented dissertation thesis titled “Scanning electron microscopy and its applications for sensitive samples” describes an instrumental and methodological development in the field of scanning electron microscopy leading to an innovative solution that could be particularly applicable in microbiological research. A summary of the history and current state of electron microscopy (EM) as a scientific imaging and analytical technique is provided in the introductory chapters. The undeniable contribution of EM in the biological and medical sciences is evidenced by many cited scientific publications. This dissertation thesis contains innovations and improvements in specimen preparation and cryogenic scanning electron microscopy (cryo-SEM) produced at the Institute of Scientific Instruments of the CAS in Brno. In particular, the new constructions of special sample holders together with methodological development in the field of microbiological sample preparation resulted in finding optimal parameters for individual processes. In the experimental part there is showed a verification of methodological procedures in the study of hydrated and electron beam sensitive specimens. Subsequent comparison of different methodological approaches on a defined microbiological system contributes to extending the interpretation of the hitherto known results. Among the microbiological strains investigated were the biofilm positive bacteria Staphylococcus epidermidis and yeasts such as Candida albicans and Candida parapsilosis, which are considered to be clinically significant because they are often involved in serious infections and especially threaten immunocompromised patients. Furthermore, the effect of the biofilm formation of Bacillus subtilis on the biodeterioration and biodegradation of poly-ε-caprolactone films was studied. The new development in low temperature cryo-SEM was employed in the research of microbes with biotechnological potential such as Cupriavidus necator and Sporobolomyces shibatanus. Key words Electron microscopy, cryo-SEM, sample preparation, microbiology, biofilm Bibliographic record HRUBANOVÁ, Kamila. Scanning electron microscopy and its applications for sensitive samples. Brno, 2019. Available: https://www.vutbr.cz/studenti/zav-prace/detail/113775. Doctoral Thesis. Brno University of Technology, Faculty of Mechanical Engineering, Institute of Physical Engineering. Supervisor Vladislav Krzyžánek. Abstrakt Předložená dizertační práce s názvem “Rastrovací elektronová mikroskopie a její aplikace pro senzitivní vzorky” pojednává o problematice rastrovací elektronové mikroskopie v kontextu instrumentálního a metodologického vývoje vedoucího k inovativnímu řešení, které je dobře aplikovatelné zejména v mikrobiologickém výzkumu. Součástí práce je rozprava o historii a současném stavu elektronové mikroskopie (EM) jakožto vědecké zobrazovací a analytické techniky, tato část se nachází v úvodních kapitolách. Nepopiratelný přínos EM v biologických a lékařských oborech je dokazován mnoha citovanými vědeckými publikacemi. Předložená dizertační práce přináší novinky z oblasti přípravy preparátů a kryogenní rastrovací elektronové mikroskopie (cryo-SEM) vyvinuté na pracovišti Ústavu přístrojové techniky AV ČR, v.v.i. v Brně. Jedná se především o návrhy a výrobu speciálních držáků vzorků a vývoj nových metodik v oblasti přípravy mikrobiologických preparátů vedoucích k nalezení optimálních parametrů jednotlivých procesů. V experimentální části se nachází ověření metodologických postupů při studiu hydratovaných a na elektronový svazek senzitivních preparátů. Následné srovnání různých přístupů na definovaném biologickém systému z oblasti mikrobiologie přispívá k rozšíření interpretace doposud známých výsledků. Mezi zkoumanými mikrobiologickými kmeny byly biofilm-pozitivní bakterie Staphylococcus epidermidis a kvasinky jako Candida albicans a Candida parapsilosis, jež jsou považovány za klinicky významné, protože se podílejí na vzniku závažných infekcí zejména u imunokompromitovaných pacientů. Dále byl studován vliv růstu biofilmu bakterie Bacillus subtilis na biodeteriorizaci a biodegradaci poly-ε-kaprolaktonových fólií. Vývoj v oblasti cryo-SEM byl aplikován ve výzkumu mikrobů s biotechnologickým potenciálem, jako jsou např. Cupriavidus necator a Sporobolomyces shibatanus. Klíčová slova Elektronová mikroskopie, cryo-SEM, příprava preparátů, mikrobiologie, biofilm Bibliografický záznam HRUBANOVÁ, Kamila. Rastrovací elektronová mikroskopie a její aplikace pro sensitivní vzorky. Brno, 2019. Dostupné také z: https://www.vutbr.cz/studenti/zav-prace/detail/113775. Dizertační práce. Vysoké učení technické v Brně, Fakulta strojního inženýrství, Ústav fyzikálního inženýrství. Vedoucí práce Vladislav Krzyžánek. Contents Contribution of the thesis ................................................................................................................. 9 List of publications ......................................................................................................................... 10 Impacted journal article .............................................................................................................. 10 Journal article ............................................................................................................................. 11 Proceedings ................................................................................................................................ 11 The engineering contribution ..................................................................................................... 14 1 State of art ................................................................................................................................... 15 1.1 Introduction to electron microscopy ..................................................................................... 15 1.1.1 Principle of the scanning electron microscope .............................................................. 18 1.1.2 Electron-optic system and electron beam control .......................................................... 18 1.1.3 Aberrations in electron microscopes ............................................................................. 21 1.2 Electron beam-specimen interactions ..................................................................................
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