Universidad Autonoma´ de Madrid Facultad de Ciencias Departamento de F´ısicade la Materia Condensada Optical and Magnetic Tweezers for Applications in Single-Molecule Biophysics and Nanotechnology Tesis elaborada por Benjamin Gollnick para optar al grado de Doctor en Biof´ısica Diciembre de 2014 Director de tesis: Dr. Fernando Moreno Herrero, cient´ıficotitular del Consejo Superior de Investigaciones Cient´ıficas(Centro Nacional de Biotecnolog´ıa(CNB{CSIC), Departamento de Estructura de Macromol´eculas) Tutor: Prof. Dr. Julio G´omezHerrero, catedr´aticode la Universidad Aut´onomade Madrid (UAM, Facultad de Ciencias, Departamento de F´ısicade la Materia Condensada) D´ıade la entrega: 2 de diciembre de 2014 D´ıade la defensa: 22 de enero de 2015 Miembros del tribunal de tesis: • Prof. Dr. Juan Jos´eS´aenzGuti´errez,catedr´aticode la Universidad Aut´onomade Madrid (UAM) | presidente • Dr. Jos´eRicardo Arias Gonz´alezde la Aleja, investigador del Instituto Madrile~no de Estudios Avanzados en Nanociencia (IMDEA Nanociencia) | secretario • Prof. Dr. Felix Ritort Farr´an,catedr´aticode la Universidad de Barcelona (UB) | vocal 1 • Dr. Carlo Manzo, investigador (research fellow) del Instituto de Ciencias Fot´onicas (ICFO), Catelldefels (Barcelona) | vocal 2 • Dr. Jorge Alegre Cebollada, investigador (jefe de grupo) del Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid | vocal 3 • Prof. Dr. Francisco Monroy Mu~noz,profesor titular de la Universidad Complutense de Madrid (UCM) | suplente 1 • Dra. Montserrat Calleja G´omez,investigadora cient´ıfica del Instituto de Microelectr´oni- ca de Madrid (IMM{CNM{CSIC) | suplente 2 Dedicated to my family and friends iv Preface \Was lange w¨ahrt,wird endlich gut." According to this popular German saying, some things may take a bit more time to get finished properly. In that sense, giving birth to this thesis truly needed some effort but has been worth the wait. It is the product of work carried out during the last six years in the field of biological physics and could not have been accomplished without the help and support from a lot of people. Some of them I would like to mention here in particular. First and foremost, I want to express my gratitude to my supervisor, Dr. Fernando Moreno, for giving me the chance to participate in the exciting process of establishing a new research laboratory. Fernando was brave enough to lend me a lot of responsibility and freedom from the very beginning, which has enabled me to gain valuable experiences I would probably not have made during a \conventional" PhD project. Although things have not always been easy, the outcome now feels even more rewarding, especially the one of Chap. 3. Thanks to Fernando, I have also been able to get to know the magnetic tweezers technique and learn some of the basics of molecular biology, which for sure will be useful for my future career. Secondly, I thank the seven members of the examination committee for assuming the duty of reviewing the manuscript and (if applicable) posing their questions on the day of defence. I should also mention Prof. Julio G´omez,who has served as my tutor at the university throughout these years. Some parts of this thesis would never have seen the light without external collaborations. I want to acknowledge Prof. Mark Dillingham and his (former) group members at the University of Bristol, in particular Neville Gilhooly, for providing us with DNA and protein samples and performing the bulk measurements shown in Chap. 5; but more importantly, for our fantastically organised mutual visits and all the fruitful discussions. I was also very lucky to spend two months in the group of Dr. Ulrich Keyser at the Cavendish Laboratory of the University of Cambridge. Since the good old times in Leipzig, Ulrich has always been a great mentor willing to share his knowledge about optical traps (and all sorts of other things). I thank him and all members of his lab for making my research stay such a pleasant one. I am especially grateful to Nadanai Laohakunakorn for letting me contribute to what turned out to be a very productive short-term project (see Chap. 4). Prof. Sandip Ghosal from Northwestern University needs to be acknowledged for figuring out the theory behind the microsphere rotations, and Prof. Dirk Aarts and Dr. Roel Dullens from the University of Oxford for preparing the dimpled colloids. At this point, I would also like to mention Hergen Brutzer, Lorenz Steinbock and Oliver Otto, all former co-workers and now friends from Germany, who have helped me through their dissertations to bring this thesis come to life. As far as internal collaborations are concerned, I am indebted to various (former) members of the groups led by Dr. Borja Ibarra and Dr. Ricardo Arias for a lot of help with all kinds of technical issues, mainly during the initial and final stages of my principal PhD project. In particular, I would like to thank Borja for both sharing his stocks of microspheres and teaching me how to functionalise them by myself. Many thanks also to Dr. El´ıasHerrero and Jos´eMor´ınfor providing me with DNA samples and buffers (and for advising me how vi to use them properly), apart from their data shown as reference curves in Chap. 3. Ricardo, together with Silvia Horme~no,needs to be mentioned as an expert in optical equipment and in setting up microscopes, which proved beneficial in the early days of construction of our optical tweezers instrument. Moreover, all of them introduced me to the challenging but yet so important task of pulling pipettes and fabricating flow cells. It has been a privilege to share the office with great colleagues, some of whom I can now consider as true friends. I would like to thank Dr. Carolina Carrasco, not only for carrying out the magnetic tweezers experiments shown in Chap. 5 and helping me translate parts of this document into Spanish, but also for being such an enjoyable person to work with. The same holds for Maru Fuentes, who has been much more than just a constant backup facilitating me the paperwork necessary to hand in this thesis, apart from proofreading parts of it and correcting my translations. Francesca Zuttion deserves a special mention, not only since she contributed some of the data described in Chap. 5, but also because she once took on with remarkable persistence a set of rather tedious measurements while I was in England. I also remember Maite Arranz as a very sympathetic computer scientist and valuable source of programming knowledge, even though we never developed the same kind of love for LabVIEW. Finally, I should acknowledge C´esarL. Pastrana for performing essential magnetic tweezers force calibrations and always being disposed to help out. To all of you and everyone else in room number B.18: I wish you the very best for your future! I also need to recognise the work done by various services and administrative departments at the CNB (almac´en, compras, mantenimiento, personal, viajes etc.) that make the life of a researcher a lot easier. In particular, I am grateful to Nicasio and (his successor) Daniel from the mechanical workshop (taller) for fabricating increasingly accurate components for our experimental setups. The technical drawings I have handed in during the last six years could make up an entire thesis chapter by themselves! I would also like to say thanks to all the (former) people from labs S.0/S.5 who I have had the pleasure to get to know, especially to Adriana, Ana, El´ıas,Mar´ıa,Rebeca and Silvia for our nice conversations during and after lunch. Last but not least, I want to thank my entire family in Germany, especially my parents, for consistently supporting me from the distance, and for always showing great interest in the things I have been doing. Here in Spain, Virtudes deserves the highest gratitude of all because without her patience and steady encouragement, especially during the most difficult times, this thesis would never have been written. I end my acknowledgements by thanking the Spanish Ministry of Education for granting me with an FPU PhD scholarship during four years of my postgraduate studies, includ- ing the costs for my two-month stay in Cambridge, and the European Research Council and the Spanish Ministry of Science and Innovation for providing my supervisor with the complementary funding I needed to initiate and terminate my research projects. Benjamin Gollnick Madrid, December 2014 Abstract Over the last few decades, continuous improvements of microscopy techniques have con- stituted one of the driving forces for the interdisciplinary field of single-molecule bio- physics { a scientific environment that bridges areas of physics, biology, chemistry and micro-/nanotechnology, amongst others. This dissertation deals with the design, construc- tion and use of two types of instruments based upon optical microscopes: optical and magnetic tweezers to manipulate individual biomolecules such as DNA or proteins in solution. First of all, the assembly of a tailor-made optical tweezers setup in an inverted microscope configuration with a single objective is discussed. Such a design provides free access to the top side of the sample stage, facilitating for instance the implementation of different flow cell types. Thanks to the development of an application-specific software, the system can exploit both video- and laser-based position detection methods { the latter via back-reflections of an additional low-power laser. We have calibrated the trap stiffness for polystyrene microspheres by spectral analysis of thermal fluctuations and viscous drag measurements. Using a micropipette as a mechanical anchor point for pulling experiments, trapping-laser- induced thermal drift effects can be minimised by active control of the objective temperature and sample stage position corrections.