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Unparalleled specificity and high sensitivity have made fluorescence microscopy an indispensable tool for life sciences. Both these aspects come from the use of light-emitting fluorophores, labeled to molecules and structures of interest. The diffraction-limited, maximum achievable resolution of traditional microscopes roughly corresponds to half the wavelength of the light used for observation. In the last decades, however, super-resolution microscopy (SRM) has been developed, which provide spatial information far beyond this diffraction limit. While different SRM techniques use different principles to achieve super resolution, most of these techniques rely on selectively switching the fluorophore emission on and off, within a diffraction-limited volume. Upon excitation, fluorophores can undergo transitions into transient dark states or become permanently photobleached. While reversible transitions may be used to achieve super resolution, they also reduce the overall emission. Characterization and modeling of photophysical dark state transitions are thus important, since they can both provide a basis for, as well as negatively affect the performance of SRM. Nevertheless, SRM has already proven valuable in biological and biomedical research, where the enhanced resolution allows for improved understanding of basic molecular mechanisms in cells and opens for future diagnostic opportunities.
This thesis presents two applications of SRM. In paper I, we used STED (stimulated emission depletion) SRM to image the disruption of beta-actin filaments in neurons infected with Streptococcus pneumoniae, suggesting a possible mechanism for neuronal death in bacterial meningitis. In paper II, the nanoscale distribution patterns of six different platelet proteins were imaged with STED to find activation-specific protein rearrangements upon co-incubation of the platelets with cancer cells. Streamlined image acquisition, analysis and classification methods were also developed, opening prospects for SRM-based minimally invasive cancer diagnosis.
Photophysical transitions of fluorophores and their implications on SRM were also studied. Cumulative photobleaching in volumetric STED imaging and how it can affect the recorded STED images was studied experimentally and verified by simulations in paper III. The effects of fluorophore transitions into transient dark states in super-resolution MINFLUX (minimal photon fluxes) were studied in paper IV. In this work, photophysical rate parameters of photo-switchable near-infrared (NIR) cyanine dyes were measured using TRAST (transient state) spectroscopy. Time evolutions of their photophysical transitions during MINFLUX localizations were then simulated, showing that fluorophore blinking can be a source of localization errors. However, from the acquired knowledge of the transient states and how they influence the localization in MINFLUX experiments, it was possible to adapt sample and excitation conditions and demonstrate MINFLUX imaging in the NIR. Thereby, it was shown that more weakly emitting and blinking NIR fluorophores can still be used in MINFLUX.