Samples of fluorescent microspheres (FluoSpheres carboxylate-modified microspheres, 48 nm actual diameter, crimson fluorescent (625/645);Life Technologies, USA) are typically employed as a first control for the setup’s performance. A solution of microspheres is diluted 1 : 5,000 with purified water, incubated on a coverslip coated with Poly-L-lysine (0.1%(w/v) in H2O;
Sigma-Aldrich, USA) for adhesion and subsequently embedded in self-prepared Mowiol. For visualizing the Point Spread Functions (PSFs), 150 nm sized reflective gold beads (BBI Solu-tions, United Kingdom) are used, following a similar incubation and embedding procedure as for the fluorescent microspheres.
Cell measurements are conducted on cells fixed in−20◦C cold methanol or in 8% PFA (para-formaldehyde, Science Services GmbH, Germany) in PBS (phosphate buffered saline, Sigma-Aldrich, USA). Immunostaining of the structures of interest is performed with Abberior STAR 635P (Abberior GmbH, Germany) or Abberior STAR RED (Abberior GmbH, Germany) as fluorescent dye coupled to a suitable secondary antibody. Depending on the application, anti-α-tubulin from mouse (T6074, Sigma-Aldrich, USA), anti-α-tubulin from rabbit (ab18251, Abcam, UK), anti-vimentin from mouse (V6389, Sigma-Aldrich, USA) or anti-PMP70 from mouse (SAB4200181, Sigma-Aldrich, USA) is chosen as primary antibody. 2% BSA (bovine serum albumin fraction V,Carl Roth GmbH, Germany) in PBS is used as blocking agent be-tween incubation steps. The sample is subsequently embedded in self-prepared Mowiol.
Actin filaments in vitro are prepared by diluting monomeric G-actin (Hypermol, Germany) to a concentration of 1µg/ml in PolyMix (Hypermol, Germany), which initiates the polymer-ization of the G-actin into the filamentous F-actin. The addition of Silicon Rhodamine (SiR, Spirochrome, Switzerland) as fluorescent dye yields a staining of the polymerized F-actin fil-aments. The solution is incubated on a coverslip for 1 min with a subsequent washing step performed with MonoMix (Hypermol, Germany). Finally, the sample is embedded in self-prepared Mowiol.
For imaging of living cells, the cells are seeded on cover glasses until the desired confluency is reached. The cell culture medium is replaced by the staining solution of 500 nmol SiR-tubulin (Spirochrome, Switzerland) in cell culture medium and the sample is incubated for three hours at 37◦C and 5 % CO2. Subsequently, the staining medium is removed and the sample is embedded on a glass slide with a well in≈50µl of DMEMgfp−2 (Evrogen, Russia).
After years of work and effort it is time to acknowledge all the help, assistance and encourage-ment I received from numerous sides and persons, whom I owe my deep gratitude. Without the scientific, non-scientific and moral support of colleagues, family and friends my PhD time would have been much less enjoyable and successful.
I’m grateful to my supervisor and primary advisor apl. Prof. Dr. Alexander Egner for giving me the opportunity to work on my PhD thesis in a very well-equipped, international laboratory group. Never tired to think about possible pathways and applications, you set the basis for many fruitful discussions leading to plenty of new approaches and ideas.
I’d like to thank Prof. Dr. Sarah Köster as my secondary advisor. Thank you for your encouragement, for your interest in the project and for providing advice, suggestions and help whenever I asked for.
I moreover thank Prof. Dr. Jörg Enderlein, Prof. Dr. Wolfram Kollatschny, Prof.
Dr. Silvio O. Rizzoliandapl. Prof. Dr. Michael Seibt for spending the time and effort to complete my thesis committee.
My sincere gratitude is owed toDr. Claudia Geislerfor supervision as well as proof-reading of the thesis. Thank you for myriads of suggestions and ideas both in the experimental work as well as in programming or analyzing data. No matter when I asked for, you always found the time to listen to any stupid problem, even if it was a mouse to be let into freedom. Thank you for your enthusiasm about little steps towards success and for making lunch breaks more enjoyable by bringing up other interesting topics than scientific ones.
Furthermore, I’d like to thankJaydev Jethwa, who - as he already did during my master’s thesis - helped me whenever high voltages came into play. Thanks to you I’m still healthy and alive and haven’t burnt down the institute neither.
High-resolution microscopy would not make sense without adequate samples. My gratitude goes to Blanka Hampe as well as Jasmin Rehman for providing fixed cells and giving suggestions and ideas on how to improve my antibody-stainings. Thank you for the very uncomplicated communication and coordination. Moreover, I owe my deep gratitude to Dr.
Kareem Solimanfor keeping the cell culture running only for me and for providing fixed and living cells whenever I asked for.
A special thanks goes to Maik Lübbecke for his help with and realization of the printed circuit board of the scan system’s control. Thank you for your time and effort in discussing possible designs, changing them following new experimental insights and finally giving a useful hand in debugging the system. Thank you for always being positive and for being interested in the outcome of my experiments.
Furthermore, I thankDr. Andreas Schönleand Dr. Jan Keller-Findeisen for providing
theMatlabcode for the PSF calculation.
I’d like to express my gratitude to all colleagues at the LLG, especially the administra-tion for a professional handling of all bureaucratic issues as well as the mechanical workshop for realizing all adapters, mounting constructions and shieldings not available on the market.
I’m grateful to all former and present members of theOptical Nanoscopy groupfor provid-ing a productive workprovid-ing environment.
ParulandDebadrita, thank you for giving a lot of insights into Indian culture and food and for always being ready to discuss scientific problems as well as the next birthday present.
Kareem, apart from keeping the cells alive, thank you for your suggestions and discussions on possible structures of interest for the different applications.
Special thanks go to René for introducing me to lab work in the beginning and for being a constant source of jokes and good humor. Thank you for always being ready to discuss any problems and for never getting tired of searching lost components in your laboratory. Thank you for many game evenings, escape and hiking adventures, which successfully distracted me from thinking about work-related problems.
I’m particularly thankful toJulia: Thank you for sharing the office and lunch breaks with me and for tolerating me in any mood. Thank you for listening and discussing problems, scientific and non-scientific ones, and for cheering me up whenever things were not going ahead like I’d wished for. You became a close friend of mine, sharing a lot of theater and concert evenings as well as trying to understand the theory and practice of folding (non-perfect, but nice-looking) origami art-work.
Not only scientific support was crucial to make this thesis become real. Without close friends, who knew to distract me whenever necessary, I wouldn’t be where I am today. I’d like to thank Hanna, Claudia and Bettina for the study time together, including a lot of card game evenings as well as outdoor adventures, and for keeping in touch also afterwards. A special thanks goes toDamaris for introducing me to math in the very beginning, encouraging me to pursue this path and moreover for being a very close and constant friend for more than a decade now - regardless of the often huge spatial distance.
I’d moreover like to thank everyone whom I had the honor to play music with, particularly Nora. Thank you for your enthusiasm, for being positive and full of energy and for updat-ing me and invitupdat-ing me to all your concerts. Special thanks also go to Jasmin, Hanna and Cornelius, with whom I’ve had the chance to rehearse for more than five years now. Thank you for never starting a rehearsal without asking how it’s going and for distracting me with Beethoven, Brahms or Schubert from any possible kind of non-music problem.
Jenny and Carsten, thank you for being such close, encouraging and supporting friends.
Thank you for taking care of me, for distracting me from work, but for also listening to me whenever problems were too big to be simply pushed aside. Jenny, thank you for all your
sup-port, smile and happiness, and thank you for proof-reading parts of this thesis in your parental leave. Thank you both for keeping in touch and for sharing unforgettable memories.
My deep gratitude is owed to those persons knowing me the longest - my parents and sib-lings. Thank you for making me the person I am, for believing in me and for trusting me to take the correct decisions even though you were not always familiar with the details of what I was doing. Thank you for always having an open ear for any not too subject-specific problem and for listening to all my complaints about things you had no chance to change. Thank you for your constant support and encouragement.
Last, but definitely not least, thank you, Francesco, first of all for your tenacity and for buying a panettone with me. Thank you for listening to and discussing scientific problems at literally any time of the day (and night) and for always being there with me and encouraging me regardless of the decisions I took and I was about to take. Thank you for believing in me especially when I didn’t and for introducing me to the Thai Chi mood, just to mention one out of an uncountable amount of things. Thank you for your unconditional support and for being the person you are.
Hiermit erkläre ich, dass ich die vorliegende Arbeit selbstständig angefertigt, nicht anderweitig zu Prüfungszwecken vorgelegt und keine anderen als die angegebenen Hilfsmittel verwendet habe. Sämtliche wissentlich verwendeten Textausschnitte, Zitate oder Inhalte anderer Verfasser wurden ausdrücklich als solche gekennzeichnet.
Britta Vinçon
1. B. Vinçon, C. Geisler, and A. Egner. Pixel hopping enables fast STED nanoscopy at low light dose. Opt. Express 28(4), 4516–4528 (2020).