|dc.description.abstracteng||Interaction and spatial organization of subcellular structures within living cells are an essential aspect of biological research, but conventional microscopy can elucidate these properties only to a ﬁnite extent as it is limited in its optical resolution by the diﬀraction barrier. In recent years, this boundary has continuously been pushed and ultimately been broken in the ﬁeld of nanoscopy. Reversible saturable optical linear ﬂuorescence transition (RESOLFT) super-resolution microscopy as one of these methods is particularly suited for nanoscopy of live cells. It utilizes the transition of ﬂuorophores between a ﬂuorescent on-state and a non-ﬂuorescent oﬀ-state and breaks the diﬀraction barrier at low light intensities in the range of 10³ W/cm². Reversibly switchable ﬂuorescent proteins (RSFPs) are established probes for RESOLFT nanoscopy. Most implementations rely on negatively switchable RSFPs, which transition to the ﬂuorescent oﬀ-state in a process driven by the same wavelength that facilitates ﬂuorescence excitation and that can be reversed with light of another wavelength. Fluorescence emission and transition to the oﬀ-state are competing processes that set a limited photon budget for RESOLFT nanoscopy. This can be circumvented by positively switchable RSFPs that have a reversed light response: their transition to the on-state is driven by their ﬂuorescence excitation wavelength, which can be subdued by another wavelength that facilitates the inverse transition. So far, the poor performance of available positively switchable RSFPs such as Padron, asFP595, and rsCherry has limited their use for nanoscopy and only few demonstrations have been published. This thesis reports the generation of Padron 2.0, which was created by multiple rounds of mutagenesis and screening from Padron.
Padron 2.0 is a signiﬁcant improvement over Padron in key aspects of ﬂuorescence state transition and is therefore particularly suited for RESOLFT nanoscopy. In contrast to Padron, Padron 2.0 displayed good expression and maturation at 37 ℃ as well as enhanced capabilities as a protein tag in live-cell applications. Most importantly, key aspects relevant for RESOLFT nanoscopy were improved. Residual ensemble oﬀ-state ﬂuorescence after switching was below 1 %, and transition to this state was more than one order of magnitude faster compared to Padron. The number of switching cycles before an ensemble of Padron 2.0 ﬂuorophores was bleached to 50 % ﬂuorescence intensity was increased more than 50 fold. Padron 1.9 and Padron 2.1 are presented as direct precursor and successor of Padron 2.0. They showed similar but less pronounced improvements and were characterized as candidates for potential niche applications. While Padron 1.9 is brighter than Padron 2.0 but more prone to switching fatigue, Padron 2.1 displays more eﬃcient transition to the oﬀ-state but is darker and tends towards oligomerization.
RESOLFT nanoscopy of Padron 2.0-labeled vimentin structures in live HeLa cells featured a lateral resolution of single ﬁlaments as low as 60 nm. In addition, the unique switching characteristics of Padron 2.0 allowed for the establishment of a novel RESOLFT imaging mode that was termed "one-step RESOLFT" in this thesis. As oﬀ-switching was more dominant than on-switching in Padron 2.0, a simpliﬁed imaging sequence with a steady-state approach and short pixel dwell times of 300 µs was demonstrated that achieved similar lateral resolution as classical RESOLFT schemes but reduced bleaching during time-lapse imaging. Padron 2.0 was shown to be the most robust and versatile positively switching RSFP to date and constitutes a promising candidate for further applications.||de