Development and characterization of new tools for advanced SMLM imaging schemes

Fluorescence microscopy has been established as a major technique in life sciences to understand biological structures and processes, as it enables high contrast imaging, provides a vast toolbox of different fluorescent markers and labeling techniques to specifically target a molecule of interest,...

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Bibliographic Details
Main Author: Balinovic, Alexander
Contributors: Endesfelder, Ulrike (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Language:English
Published: Philipps-Universität Marburg 2024
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Summary:Fluorescence microscopy has been established as a major technique in life sciences to understand biological structures and processes, as it enables high contrast imaging, provides a vast toolbox of different fluorescent markers and labeling techniques to specifically target a molecule of interest, and allows for live- and fixed cell compatible imaging experiments. To resolve structures smaller than 200 nm, which corresponds to the diffraction limit of light, more sophisticated methods are required. Although techniques such as electron microscopy grant the necessary resolution they do not provide the contrast and live-cell compatibility yielded by fluorescence microscopy. Thus, to circumvent the diffraction limit of light and to utilize the aforementioned advantages of fluorescence microscopy, super-resolution microscopy (SRM) techniques were developed by designing and applying sophisticated illumination patterns and fluorophores with controllable photophysics. single-molecule localization microscopy (SMLM) methods, such as photoactivated localization microscopy (PALM), direct stochastic optical reconstruction microscopy (dSTORM) or points accumulation for imaging in nanoscale topography (PAINT), have greatly expanded the toolbox of SRM and have been extensively used to qualitatively and quantitatively analyze the spatial organization and molecular dynamics of intracellular structures and processes. Nevertheless, the application of SMLM is also accompanied by a few drawbacks that have to be considered. The quality of SMLM images and thus the biological results can be impaired by e.g. the usage of high laser irradiations that are required in SMLM experiments, which can lead to phototoxic damage in the biological sample, by heterogeneities in the illumination pattern, and by sample drift occurring during image acquisition. These issues raise the demand for tools for robust and precise drift correction, and for determining the irradiation power and illumination heterogeneities directly at the sample level. Thus, in chapter 2, novel tools and strategies to tackle these issues were developed. Precise and robust drift correction based on fiducial markers requires the use of monodisperse fluorescent fiducials displaying a stable fluorescent signal during the SMLM image acquisition that is significantly brighter than the signal of the target fluorophore. Hence, the performance of different commonly-used fiducial markers was first evaluated and then a new strategy for fiducial based drift correction was established. Commonly-used fiducial markers, such as TetraSpeck beads or gold nanoparticles either photobleached or displayed signal fluctuations during image acquisitions, or formed aggregates and were not much brighter than the target fluorophore and thus yielded unprecise drift correction results. Therefore, a new strategy was introduced using fluorescent beads as fiducials that are coated with a fluorescent dye, which is spectrally more red-shifted than the read-out channel of the target fluorophore. This low efficiency excitation and the high number of dye molecules the fiducials are coated with resulted in a sufficiently bright and more stable fluorescent signal and thus a more precise drift correction. This strategy was then further expanded by designing a probe that can serve as a fiducial for drift correction but can also as a fluorescent brightness standard to robustly determine illumination heterogeneities and irradiation doses at the sample level due to its defined structure and number of dye molecules attached to it. To ensure both a stable fluorescence signal and a defined fiducial structure and number of fluorophores, the concepts of the DNA origami technique and DNA-points accumulation for imaging in nanoscale topography (DNA-PAINT) were combined by using a DNA origami construct with a defined number of binding sites and transiently labeling it with a fluorescent dye bound to a short DNA strand that has a complementary sequence to the binding site on the DNA origami. By modulating the dye concentration and imaging buffer salinity, it was possible to ensure a quasi-constant and complete labeling of the DNA origami resulting in a stable fluorescent signal. Finally, in chapter 3 the SMLM method single-particle tracking photoactivated localization microscopy (sptPALM) was utilized and established as a powerful technique to elucidate the molecular dynamics and interactions of single proteins and protein complexes in living cells, with the type III secretion system (T3SS) in Yersinia enterocolitica and its components being the main focus. The T3SS is a syringe-shaped multi-protein complex deployed by many pathogenic bacteria to interact and manipulate host cells by secreting molecular toxins, called effector proteins. Although the overall structure of the T3SS is already well-studied, its regulation and function are so far only scarcely understood. sptPALM was used to study two subcomplexes of the T3SS in living Y. enterocolitica cells: the cytosolic interaction between the sorting platform and the effector proteins, and the pH-dependent diffusion and assembly of the anchor protein SctD. By monitoring the diffusion profile of the sorting platform core component SctQ in presence and absence of effector proteins and under both nonsecreting and secreting conditions, it was possible to show the interaction between the sorting platform and effector proteins, which due to the transient nature of the interaction proved to be challenging to detect using conventional protein interaction studies. Furthermore, it was shown that the presence of effectors and the secreting conditions influence the overall composition of the sorting platform. The diffusion profile of the inner membrane protein SctD revealed a partial dissociation of SctD at low external pH, which could be fully restored by recovery of neutral external pH. This observation strongly indicates a regulatory function, as the partial dissociation of SctD at low pH would prevent effector secretion in e.g. the stomach, while recovery to neutral pH would enable a fast activation of the secretion machinery within the targeted infection region. As this mechanism was only observed in gastrointestinal pathogens, such as Y. enterocolitica and S. flexneri and not in pathogenic bacteria with other infection routes, such as P. aeruginosa reinforced the hypothesis that this mechanism provides gastrointestinal pathogenic bacteria with a physiological advantage to conserve energy and temporary suppress effector secretion at low external pH.
Physical Description:229 Pages
DOI:10.17192/z2024.0103