title: Development and characterization of new tools for advanced SMLM imaging schemes
title: Entwicklung und Charakterisierung von neuen Werkzeugen für fortgeschrittene Einzelmolekül-Lokalisationsmikroskopie
titleType: AlternativeTitle
Creators: name: Balinovic, Alexander
Publisher: name: Philipps-Universität Marburg
Year: 2024
Subjects: Driftkorrektur
Fluoreszenzmikroskopie
Fluorescence
Drift correction
Injektisom
Super-resolution microscopy
SMLM
Yersinia enterocolitica
DNA-Origami
Injectisome
sptPALM
Einzelmolekül-Lokalisationsmikroskopie
Life sciences
Biowissenschaften, Biologie
Mikrobiologie
Tracking
Beads

description: 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.