cam a22 4 4500 555238261 CHVBK 20201202100938.0 190203s2018 sz a m 00 0deng d urn:nbn:ch:bel-1576928 urn (SNL)991017944813103976 (SNL)991018070824003976 (NEBIS)011340831 (Sz)991017944813103976 (Sz)991018070824003976 Sz ger rda eng eng ger 570 23sdnb Mohr Manuel A. 1989- (DE-588)1175406570 Verfasser aut Tools and techniques for single cell applications in development and neurobiology presented by Manuel Alexander Mohr Zurich 2018 xviii, 167 Seiten Illustrationen 30 cm Dissertation No. 25395 ETH Zurich, 2018 Literaturverzeichnis application/pdf Fluorescence microscopy of genetically encoded fluorescent proteins (FPs) and biosensors has transformed modern biological research a phenomenon often referred to as the "fluorescent protein revolution". From the very beginning of this revolution, the discovery and engineering of new protein probes and the development of new microscopy modalities have mutually enabled each other. Prominently, breaking the fundamental diffraction limit of light in various super-resolution modalities was enabled by the discovery and engineering of photomodulatable FPs such as the photoactivatable paGFP or the photoconvertible FPs Dendra2 or EosFP. While photoconvertible FPs (pcFPs) are formidable markers for super-resolution microscopy and highlighting of structures or cells for pulse-chase-type experiments, they suffer from some key drawbacks for applications in complex, sensitive biological tissues. The near-ultraviolet (UV) light needed for efficient photoconversion cannot be axially confined inside tissue volumes, has limited tissue penetration and is phototoxic to light sensitive samples such as developing embryos. Primed conversion elegantly overcomes this requirement for near-UV light and can be axially confined to single cells in three dimensional tissues. However, this process has been limited to a single FP and remained mechanistically elusive, preventing protein engineering of probes as well as many biological applications. Following the introduction, in the second part of this thesis I describe my efforts to advance both the infrastructure and protein probes for primed conversion. I provide a detailed protocol for the simple, safe, and reproducible implementation of primed conversion into a commercial confocal laser scanning microscope (CLSM). Further, I uncover the mechanistic details underlying primed conversion and use this knowledge to engineer a host of pcFPs with improved characteristics for various primed conversion applications. I establish the first ever multi-color photoactvation localization microscopy (PALM)scheme using two spectrally identical variants of mEos2, separating them solely based on their mode of photoconversion. Further, I provide a semiautomatic platform to use primed conversion of single blastomeres in a developing mouse embryo to computationally reorient the embryo and correct for the rapid movement and rotation, that would otherwise lead to the exclusion of the respective embryo from analysis. In the third part of this thesis I talk about a new class of genetically encoded Calcium ion (Ca2+) indicators GECIs, that we developed for two-photon (2P) imaging using cheap and powerful alternatives to the commonly used Titanium Sapphire (Ti::Sapph) lasers. We optimize several variants of the best in class GECIs of the GCaMP-series to excite efficiently at the center wavelength of non-tunable 1030nm Ytterbium-doped optical fiber lasers (YbFLs). This spectral shift allows for traditional 2P imaging using lasers of drastically reduced costs as well as kHz frame-rate imaging of Ca2+-dynamics using swept line angular projection microscopy (SLAPMi). Furthermore, I propose these new GECIs to enable simultaneous dual-color Ca2+-imaging together with existing red GECIs such as jRGECO1a. Englischer Text mit englischer und deutscher Zusammenfassung EXPERIMENTE AN LEBENDEN ZELLEN, METHODEN UND TECHNIKEN ger (ETHUDK)000021350 ethudk FLUORESZIERENDE PROTEINE ger (ETHUDK)000063709 ethudk KONFOKALE MIKROSKOPIE (OPTIK) ger (ETHUDK)000052777 ethudk PHOTOREGULATION + PHOTOAKTIVIERUNG + PHOTOINAKTIVIERUNG + PHOTOREAKTIVIERUNG (BIOPHYSIK) ger (ETHUDK)000022941 ethudk Hochschulschrift (DE-588)4113937-9 gnd-content Hochschulschrift gnd-content sb 2019/03 570 zh snl-sb xehelv xediss 2020 570 snl ehelv u KONFOKALE MIKROSKOPIE (OPTIK) ger 535.8,12 nebis E1 u FLUORESZIERENDE PROTEINE ger 577.112.8*FLUORES nebis E1 u PHOTOREGULATION + PHOTOAKTIVIERUNG + PHOTOINAKTIVIERUNG + PHOTOREAKTIVIERUNG (BIOPHYSIK) ger 577.344.2 nebis E1 u EXPERIMENTE AN LEBENDEN ZELLEN, METHODEN UND TECHNIKEN ger 576.086.8 nebis E1 u CONFOCAL MICROSCOPY (OPTICS) eng 535.8,12 nebis E1 u MICROSCOPIE CONFOCALE (OPTIQUE) fre 535.8,12 nebis E1 u FLUORESCENT PROTEINS eng 577.112.8*FLUORES nebis E1 u PROTÉINES FLUORESCENTES fre 577.112.8*FLUORES nebis E1 u PHOTOREGULATION + PHOTOACTIVATION + PHOTOINACTIVATION + PHOTOREACTIVATION (BIOPHYSICS) eng 577.344.2 nebis E1 u PHOTORÉACTIVATION + PHOTOINACTIVATION + PHOTOACTIVATION + PHOTORÉGULATION (BIOPHYSIQUE) fre 577.344.2 nebis E1 u EXPÉRIENCES SUR CELLULES ET TISSUS VIVANTES, MÉTHODES ET TECHNIQUES fre 576.086.8 nebis E1 u EXPERIMENTS ON LIVING CELLS, METHODS AND TECHNIQUES eng 576.086.8 nebis E1 Eidgenössische Technische Hochschule Zürich (DE-588)2023928-2 Grad-verleihende Institution dgg SNL NB001 NB999 - chb99 - https://opac.admin.ch/toc/toc1902044404.pdf Inhaltsverzeichnis BK020300 XK020000 XK020000 031 E01-20190121 SNL NB001 NB001 Nbq 119206 NB10010 NEBIS E16 E16 RH Diss ETH 25395 SNL 100 1- Mohr Manuel A. 1989- (DE-588)1175406570 Verfasser aut SNL 856 41 https://opac.admin.ch/toc/toc1902044404.pdf Inhaltsverzeichnis SNL 710 2- Eidgenössische Technische Hochschule Zürich (DE-588)2023928-2 Grad-verleihende Institution dgg SNL 100 1- Mohr Manuel A. Verfasser aut SNL 856 40 http://hdl.handle.net/20.500.11850/301753 SNL 856 4- https://nbn-resolving.org/urn:nbn:ch:bel-1576928 SNL 856 42 http://www.e-helvetica.nb.admin.ch/directAccess?callnumber=bel-1576928 NEBIS 100 1- Mohr Manuel A. 1989- (DE-588)1175406570 Verfasser aut NEBIS 700 1- Pantazis Periklis 1975- (DE-588)129827312 Akademischer Betreuer dgs NEBIS 700 1- Schreiter Eric R. Akademischer Betreuer dgs