@phdthesis{Trinks2024,
  author    = {Trinks, Nora Isabel},
  title     = {Super-resolution fluorescence microscopic visualization and analysis of interactions between human immune cells and \(Aspergillus\) \(fumigatus\)},
  doi       = {10.25972/OPUS-26640},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-266407},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {The mold Aspergillus fumigatus (A. fumigatus) is known as human pathogen and can cause life-threatening infections in humans with a weakened immune system. This is a known complication in patients receiving glucocorticoids, e.g. after hematopoietic stem cell transplantation or solid organ transplantation. Although research in the field of immune cell/fungus interaction has discovered key strategies how immune cells fight against infectious fungi, our knowledge is still incomplete. In order to develop effective treatment options against fungal infections, a detailed understanding of their interactions is crucial. Thus, visualization of immune cell and fungus is an excellent approach to gain further knowledge. For a detailed view of such interaction processes, a high optical resolution on nanometer scale is required. There is a variety of super resolution microscopy techniques, enabling fluorescence imaging beyond the diffraction limit. This work combines the use of three complementary super resolution microscopy techniques, in order to study immune cell/fungus interaction from different points of view. Aim of this work is the introduction of the recently invented imaging technique named expansion microscopy (ExM) for the study of immune cell/fungus interactions. The core aspect of this method is the physical magnification of the specimen, which increases the distance between protein structures that are close to each other and which can therefore be imaged separately. The simultaneous magnification of primary human natural killer (NK) cells and A. fumigatus hyphae was established in this work using ExM. Reorganization of cytoskeletal components of interacting NK cells was demonstrated here, by expansion of the immunological synapse (IS), formed between NK cells and A. fumigatus. In addition, reorganization of the microtubule-organizing center (MTOC) towards fungal hyphae and an accumulation of actin at the IS has been observed. Furthermore, ExM has been used to visualize lytic granules of NK cells after degranulation. After magnification of the specimen, lysosome associated protein 1 (LAMP1) was shown to surround perforin. In absence of the plasma membrane-exposed degranulation marker LAMP1, a "ring-shaped" structure was often observed for fluorescently labeled perforin. Volume calculation of lytic granules demonstrated the benefit of ExM. Compared to pre-expansion images, analyses of post-expansion images showed two volume distributions for degranulated and non-degranulated NK cells. In addition, this work emphasizes the importance of determining the expansion factor for a structure in each species, as variations of expansion factors have been observed. This factor, as well as possible sample distortions should be considered, when ExM is used in order to analyze the interaction between two species. A second focus of this work is the visualization of a chimeric antigen receptor (CAR), targeting an epitope on the cell wall of A. fumigatus. Structured illumination microscopy (SIM) revealed that the CAR is part of the immunological synapse of primary human CAR T cells and CAR-NK-92 cells. At the interaction site, an accumulation of the CAR was observed, as well as the presence of perforin. CAR accumulation at fungal hyphae was further demonstrated by automated live cell imaging of interacting CAR-NK-92 cells, expressing a fluorescent fusion protein. Additionally, the use of direct stochastic optical reconstruction microscopy (dSTORM) gave first insights in CAR expression levels on the basal membrane of CAR-NK-92 cells, with single molecule sensitivity. CAR cluster analyses displayed a heterogeneous CAR density on the basal membrane of transfected NK 92 cells. In summary, this work provides insights into the application of ExM for studying the interaction of primary human NK cells and A. fumigatus for the first time. Furthermore, this thesis presents first insights regarding the characterization of an A. fumigatus-targeting CAR, by applying super-resolution fluorescence microscopy, like SIM and dSTORM.},
  subject      = {Mikroskopie},
  language  = {en}
}
@phdthesis{Nordblom2023,
  author    = {Nordblom, Noah Frieder},
  title     = {Synthese und Evaluation von Gephyrinsonden f{\"u}r hochaufl{\"o}sende Mikroskopieverfahren},
  doi       = {10.25972/OPUS-30230},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-302300},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {This decade saw the development of new high-end light microscopy approaches. These technologies are increasingly used to expand our understanding of cellular function and the molecular mechanisms of life and disease. The precision of state-of-the-art super resolution microscopy is limited by the properties of the applied fluorescent label. Here I describe the synthesis and evaluation of new functional fluorescent probes that specifically stain gephyrin, universal marker of the neuronal inhibitory post-synapse. Selected probe precursor peptides were synthesised using solid phase peptide synthesis and conjugated with selected super resolution capable fluorescent dyes. Identity and purity were defined using chromatography and mass spectrometric methods. To probe the target specificity of the resulting probe variants in cellular context, a high-throughput assay was established. The established semi-automated and parallel workflow was used for the evaluation of three selected probes by defining their co-localization with the expressed fluorescent target protein. My work provided NN1Dc and established the probe as a visualisation tool for essentially background-free visualisation of the synaptic marker protein gephyrin in a cellular context. Furthermore, NN1DA became part of a toolbox for studying the inhibitory synapse ultrastructure and brain connectivity and turned out useful for the development of a label-free, high-throughput protein interaction quantification assay.},
  subject      = {Fluoreszenzmikroskopie},
  language  = {en}
}
@phdthesis{Niehoerster2022,
  author    = {Nieh{\"o}rster, Thomas},
  title     = {Spektral aufgel{\"o}ste Fluoreszenzlebensdauer-Mikroskopie mit vielen Farben},
  doi       = {10.25972/OPUS-29657},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-296573},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {Die Fluoreszenzmikroskopie ist eine vielseitig einsetzbare Untersuchungsmethode f{\"u}r biologische Proben, bei der Biomolek{\"u}le selektiv mit Fluoreszenzfarbstoffen markiert werden, um sie dann mit sehr gutem Kontrast abzubilden. Dies ist auch mit mehreren verschiedenartigen Zielmolek{\"u}len gleichzeitig m{\"o}glich, wobei {\"u}blicherweise verschiedene Farbstoffe eingesetzt werden, die {\"u}ber ihre Spektren unterschieden werden k{\"o}nnen. Um die Anzahl gleichzeitig verwendbarer F{\"a}rbungen zu maximieren, wird in dieser Arbeit zus{\"a}tzlich zur spektralen Information auch das zeitliche Abklingverhalten der Fluoreszenzfarbstoffe mittels spektral aufgel{\"o}ster Fluoreszenzlebensdauer-Mikroskopie (spectrally resolved fluorescence lifetime imaging microscopy, sFLIM) vermessen. Dazu wird die Probe in einem Konfokalmikroskop von drei abwechselnd gepulsten Lasern mit Wellenl{\"a}ngen von 485 nm, 532nm und 640nm angeregt. Die Detektion des Fluoreszenzlichtes erfolgt mit einer hohen spektralen Aufl{\"o}sung von 32 Kan{\"a}len und gleichzeitig mit sehr hoher zeitlicher Aufl{\"o}sung von einigen Picosekunden. Damit wird zu jedem detektierten Fluoreszenzphoton der Anregungslaser, der spektrale Kanal und die Ankunftszeit registriert. Diese detaillierte multidimensionale Information wird von einem Pattern-Matching-Algorithmus ausgewertet, der das Fluoreszenzsignal mit zuvor erstellten Referenzpattern der einzelnen Farbstoffe vergleicht. Der Algorithmus bestimmt so f{\"u}r jedes Pixel die Beitr{\"a}ge der einzelnen Farbstoffe. Mit dieser Technik konnten pro Anregungslaser f{\"u}nf verschiedene F{\"a}rbungen gleichzeitig dargestellt werden, also theoretisch insgesamt 15 F{\"a}rbungen. In der Praxis konnten mit allen drei Lasern zusammen insgesamt neun F{\"a}rbungen abgebildet werden, wobei die Anzahl der Farben vor allem durch die anspruchsvolle Probenvorbereitung limitiert war. In anderen Versuchen konnte die sehr hohe Sensitivit{\"a}t des sFLIM-Systems genutzt werden, um verschiedene Zielmolek{\"u}le voneinander zu unterscheiden, obwohl sie alle mit demselben Farbstoff markiert waren. Dies war m{\"o}glich, weil sich die Fluoreszenzeigenschaften eines Farbstoffmolek{\"u}ls geringf{\"u}gig in Abh{\"a}ngigkeit von seiner Umgebung {\"a}ndern. Weiterhin konnte die sFLIM-Technik mit der hochaufl{\"o}senden STED-Mikroskopie (STED: stimulated emission depletion) kombiniert werden, um so hochaufgel{\"o}ste zweifarbige Bilder zu erzeugen, wobei nur ein einziger gemeinsamer STED-Laser ben{\"o}tigt wurde. Die gleichzeitige Erfassung von mehreren photophysikalischen Messgr{\"o}ßen sowie deren Auswertung durch den Pattern-Matching-Algorithmus erm{\"o}glichten somit die Entwicklung von neuen Methoden der Fluoreszenzmikroskopie f{\"u}r Mehrfachf{\"a}rbungen.},
  subject      = {Fluoreszenzmikroskopie},
  language  = {de}
}
@phdthesis{Oberndorfer2022,
  author    = {Oberndorfer, Florian},
  title     = {Photoluminescence and Raman spectroscopy of doped nanomaterials},
  doi       = {10.25972/OPUS-27854},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-278540},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {This thesis includes measurements that were recorded by cooperation partners. The EPR spec- trosa mentioned in section 5.2 were recorded by Michael Auth from the Dyakonov Group (Ex- perimental Physics VI, Julius-Maximilians-Universit{\"a}t, W{\"u}rzburg). The TREFISH experiments and transient absorption in section 5.4 spectra were performed by Jašinskas et al. from the V. Gulbi- nas group (Center for Physical Sciences and Technology, Vilnius, Lithuania). This dissertation investigated the interactions of semiconducting single-walled carbon nanotubes (SWNTs) of (6,5) chirality with their environment. Shear-mixing provided high-quality SWNT sus- pensions, which was complemented by various film preparation techniques. These techniques were in turn used to prepare heterostructures with MoS2 and hBN, which were examined with a newly constructed photoluminescence microscope specifically for this purpose. Finally, the change of spectral properties of SWNTs upon doping was investigated in more detail, as well as the behaviour of charge carriers in the tubes themselves. To optimise the SWNT sample preparation techniques that supplied the other experiments, the sample quality of shear-mixed preparations was compared with that of sonicated samples. It was found that the quantum efficiency of sheared suspensions exceeds that of sonicated suspensions as soon as the sonication time exceeds 30 min. The higher PLQY is due to the lower defect concentration in shear-mixed samples. Via transient absorption, a mean lifetime of 17.3 ps and a mean distance between defects of 192.1 nm could be determined. Furthermore, it was found that the increased efficiency of horn sonication is probably not only due to higher shear forces acting on the SWNT bundles but also that the shortening of PFO-BPy strands plays a significant role. Sonication of very long polymer strands significantly increased their effectiveness in shear mixing. While previous approaches could only achieve very low concentrations of SWNTs in suspensions, pre-sonicated polymer yielded results which were comparable with much shorter PFO-BPy batches. Reference experiments also showed that different aggregation processes are relevant during production and further processing. Initial reprocessing of carbon nanotube raw material requires 7 h sonication time and over 24 h shear mixing before no increase in carbon nano concentration is detectable. However, only a few minutes of sonication or shear mixing are required when reprocessing the residue produced during the separation of the slurry. This discrepancy indicates that different aggregates are present, with markedly different aggregation properties. To study low-dimensional heterostructures, a PL microscope was set up with the ability to ob- serve single SWNTs as well as monolayers of other low-dimensional systems. Furthermore, sam- ples were prepared which bring single SWNTs into contact with 2D materials such as h-BN andMoS2 layers and the changes in the photoluminescence spectrum were documented. For h-BN, it was observed whether previous methods for depositing SWNTs could be transferred for photo- luminescence spectroscopy. SWNTs were successfully deposited on monolayers via a modified drip coating, with the limitation that SWNTs aggregate more at the edges of the monolayers. Upon contact of SWNTs with MoS2, significant changes in the emission properties of the mono- layers were observed. The fluorescence, which was mainly dominated by excitons, was shifted towards trion emission. Reference experiments excluded PFO-BPy and toluene as potential causes. Based on the change in the emission behaviour of MoS2, the most plausible explanation is a photoinduced charge transfer leading to delocalised charge carriers on MoS2. In contrast, on SWNTs, the introduction of additional charges would constitute a quenching centre, which would quench their PL emission, making them undetectable in the PL image. In the last chapter, the electronic properties of doped SWNTs and the behaviour of charge carri- ers inside the tubes should be investigated. First, the change in the conductivity of SWNT films with increasing doping levels was docu- mented. The resistance of the films drops drastically at minimum doping. After the initial in- troduction of charges, the resistance drops with increasing dopant concentration according to a double logarithmic curve. The initial drop could be due to a reduction of contact resistances within the SWNT network film, but this could not be further investigated within the scope of this PhD thesis. In cooperation with Andreas Sperlich and Michael Auth, the spin concentration of SWNTs at different doping levels was determined. The obtained concentrations were compared with the carrier concentrations determined from PL and absorption spectra. At low spin densities, good agreement with previous models was found. Furthermore, the presence of isolated spins strongly suggests a localised charge carrier distribution at temperatures around 10 K. When the charge density is increased, the spin density deviates significantly from the charge carrier con- centration. This discrepancy is attributed to the increasing delocalisation of charge carriers at high charge densities and the interactions of neighbouring spins. These results strongly indicate the existence of localised charge carriers in SWNTs at low temperatures. Next, the effect of doping on the Raman spectra of SWNT suspensions was investigated. In gen- eral, doping is expected to reduce the intensity of the Raman bands, i.e. a consequence of the reduced resonance gain due to bleaching of the S2 transition. However, similar to the resistivity measurements, the oscillator strength of the G+ band drops sharply in the first doping steps. It was also found that the G+ band decreases more than would be expected due to loss of reso- nance condition. Furthermore, the G- is bleached faster than the G+ band. All these anomalies suggest that resonance enhancement is not the only relevant effect. Another much faster deac- tivation path for the excitons may be introduced by doping. This would leave less time for the scattering process to occur and reduce the oscillator strength of the Raman bands. In cooperation with Vidmantas et al., the photoinduced charge carrier behaviour of SWNT/PCBM films was investigated. The required films were prepared by drop coating. The SWNT suspen- sions required for this were obtained from sheared SWNT preparations. Using transient absorp- tion and TREFISH, a number of charge transfer effects were identified and their dynamics in- vestigated: the recombination of neutral excitons (< 50 ps), the electron transfer from carbon nanotubes to PCBM molecules (< 1 ps), the decay of charge-transfer excitons (∼200 ps), the recombination of charge carriers between charge-transfer excitons (1 ns to 4 ns) and finally the propagation through the SWNT network (∼20 ns)},
  subject      = {Einwandige Kohlenstoff-Nanor{\"o}hre},
  language  = {en}
}
@phdthesis{BathePeters2022,
  author    = {Bathe-Peters, Marc},
  title     = {Spectroscopic approaches for the localization and dynamics of β\(_1\)- and β\(_2\)-adrenergic receptors in cardiomyocytes},
  doi       = {10.25972/OPUS-25812},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-258126},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {In the heart the β\(_1\)-adrenergic receptor (AR) and the β\(_2\)-AR, two prototypical G protein-coupled receptors (GPCRs), are both activated by the same hormones, namely adrenaline and noradrenaline. Both receptors couple to stimulatory G\(_s\) proteins, mediate an increase in cyclic adenosine monophosphate (cAMP) and influence the contractility and frequency of the heart upon stimulation. However, activation of the β\(_1\)-AR, not the β\(_2\)-AR, lead to other additional effects, such as changes in gene transcription resulting in cardiac hypertrophy, leading to speculations on how distinct effects can arise from receptors coupled to the same downstream signaling pathway. In this thesis the question of whether this distinct behavior may originate from a differential localization of these two receptors in adult cardiomyocytes is addressed. Therefore, fluorescence spectroscopy tools are developed and implemented in order to elucidate the presence and dynamics of these endogenous receptors at the outer plasma membrane as well as on the T-tubular network of intact adult cardiomyocytes. This allows the visualization of confined localization and diffusion of the β\(_2\)-AR to the T-tubular network at endogenous expression. In contrast, the β\(_1\)-AR is found diffusing at both the outer plasma membrane and the T-tubules. Upon overexpression of the β\(_2\)-AR in adult transgenic cardiomyocytes, the receptors experience a loss of this compartmentalization and are also found at the cell surface. These data suggest that distinct signaling and functional effects can be controlled by specific cell surface targeting of the receptor subtypes. The tools at the basis of this thesis work are a fluorescent adrenergic antagonist in combination of fluorescence fluctuation spectroscopy to monitor the localization and dynamics of the lowly expressed adrenergic receptors. Along the way to optimizing these approaches, I worked on combining widefield and confocal imaging in one setup, as well as implementing a stable autofocus mechanism using electrically tunable lenses.},
  subject      = {G-Protein gekoppelte Rezeptoren},
  language  = {en}
}
@phdthesis{Gentzsch2021,
  author    = {Gentzsch, Christian},
  title     = {Molecular Imaging of Opioid Receptors and Butyrylcholinesterase with Selective, Tailored Probes Using Positron Emission Tomography and Fluorescence Microscopy},
  doi       = {10.25972/OPUS-24752},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-247529},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {The present thesis concerns the molecular imaging of opioid receptors and human butyrylcholinesterase with the aid of tailored probes, which are suitable for the respective applied imaging techniques. The first part focusses on imaging of opioid receptors with selective probes using total internal reflection- and single molecule fluorescence microscopy. Design and synthesis of the ligands are presented and their pharmacological characterization and application in microscopy experiments are shown. The second part of this thesis focused on the development of 18F-labeled, selective radiotracers for imaging of butyrylcholinesterase via positron emission tomography. The design and synthesis of each a reversible and pseudoirreversible 18F-labeled tracer are presented. After evaluation of the binding properties of each tracer, their initial application in ex vivo autoradiography- and preliminary in vivo microPET studies is described and analyzed.},
  subject      = {Fluoreszenzmikroskopie},
  language  = {en}
}
@phdthesis{Eiring2021,
  author    = {Eiring, Patrick},
  title     = {Super-resolution microscopy of plasma membrane receptors},
  doi       = {10.25972/OPUS-25004},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-250048},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Plasma membrane receptors are the most crucial and most commonly studied components of cells, since they not only ensure communication between the extracellular space and cells, but are also responsible for the regulation of cell cycle and cell division. The composition of the surface receptors, the so-called "Receptome", differs and is characteristic for certain cell types. Due to their significance, receptors have been important target structures for diagnostic and therapy in cancer medicine and often show aberrant expression patterns in various cancers compared to healthy cells. However, these aberrations can also be exploited and targeted by different medical approaches, as in the case of personalized immunotherapy. In addition, advances in modern fluorescence microscopy by so-called single molecule techniques allow for unprecedented sensitive visualization and quantification of molecules with an attainable spatial resolution of 10-20 nm, allowing for the detection of both stoichiometric and expression density differences. In this work, the single molecule sensitive method dSTORM was applied to quantify the receptor composition of various cell lines as well as in primary samples obtained from patients with hematologic malignancies. The focus of this work lies on artefact free quantification, stoichiometric analyses of oligomerization states and co localization analyses of membrane receptors. Basic requirements for the quantification of receptors are dyes with good photoswitching properties and labels that specifically mark the target structure without generating background through non-specific binding. To ensure this, antibodies with a predefined DOL (degree of labeling) were used, which are also standard in flow cytometry. First background reduction protocols were established on cell lines prior analyses in primary patient samples. Quantitative analyses showed clear expression differences between the cell lines and the patient cells, but also between individual patients. An important component of this work is the ability to detect the oligomerization states of receptors, which enables a more accurate quantification of membrane receptor densities compared to standard flow cytometry. It also provides information about the activation of a certain receptor, for example of FLT3, a tyrosine kinase, dimerizing upon activation. For this purpose, different well-known monomers and dimers were compared to distinguish the typical localization statistics of single bound antibodies from two or more antibodies that are in proximity. Further experiments as well as co localization analyses proved that antibodies can bind to closely adjacent epitopes despite their size. These analytical methods were subsequently applied for quantification and visualization of receptors in two clinically relevant examples. Firstly, various therapeutically relevant receptors such as CD38, BCMA and SLAMF7 for multiple myeloma, a malignant disease of plasma cells, were analyzed and quantified on patient cells. Furthermore, the influence of TP53 and KRAS mutations on receptor expression levels was investigated using the multiple myeloma cell lines OPM2 and AMO1, showing clear differences in certain receptor quantities. Secondly, FLT3 which is a therapeutic target receptor for acute myeloid leukemia, was quantified and stoichiometrically analyzed on both cell lines and patient cells. In addition, cells that have developed resistance against midostaurin were compared with cells that still respond to this type I tyrosine-kinase-inhibitor for their FLT3 receptor expression and oligomerization state.},
  subject      = {Fluoreszenzmikroskopie},
  language  = {en}
}
@phdthesis{Dannhaeuser2021,
  author    = {Dannh{\"a}user, Sven},
  title     = {Function of the Drosophila adhesion-GPCR Latrophilin/CIRL in nociception and neuropathy},
  doi       = {10.25972/OPUS-20158},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-201580},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Touch sensation is the ability to perceive mechanical cues which is required for essential behaviors. These encompass the avoidance of tissue damage, environmental perception, and social interaction but also proprioception and hearing. Therefore research on receptors that convert mechanical stimuli into electrical signals in sensory neurons remains a topical research focus. However, the underlying molecular mechanisms for mechano-metabotropic signal transduction are largely unknown, despite the vital role of mechanosensation in all corners of physiology. Being a large family with over 30 mammalian members, adhesion-type G protein-coupled receptors (aGPCRs) operate in a vast range of physiological processes. Correspondingly, diverse human diseases, such as developmental disorders, defects of the nervous system, allergies and cancer are associated with these receptor family. Several aGPCRs have recently been linked to mechanosensitive functions suggesting, that processing of mechanical stimuli may be a common feature of this receptor family - not only in classical mechanosensory structures. This project employed Drosophila melanogaster as the candidate to analyze the aGPCR Latrophilin/dCIRL function in mechanical nociception in vivo. To this end, we focused on larval sensory neurons and investigated molecular mechanisms of dCIRL activity using noxious mechanical stimuli in combination with optogenetic tools to manipulate second messenger pathways. In addition, we made use of a neuropathy model to test for an involvement of aGPCR signaling in the malfunctioning peripheral nervous system. To do so, this study investigated and characterized nocifensive behavior in dCirl null mutants (dCirlKO) and employed genetically targeted RNA-interference (RNAi) to cell-specifically manipulate nociceptive function. The results revealed that dCirl is transcribed in type II class IV peripheral sensory neurons - a cell type that is structurally similar to mammalian nociceptors and detects different nociceptive sensory modalities. Furthermore, dCirlKO larvae showed increased nocifensive behavior which can be rescued in cell specific reexpression experiments. Expression of bPAC (bacterial photoactivatable adenylate cyclase) in these nociceptive neurons enabled us to investigate an intracellular signaling cascade of dCIRL function provoked by light-induced elevation of cAMP. Here, the findings demonstrated that dCIRL operates as a down-regulator of nocifensive behavior by modulating nociceptive neurons. Given the clinical relevance of this results, dCirl function was tested in a chemically induced neuropathy model where it was shown that cell specific overexpression of dCirl rescued nocifensive behavior but not nociceptor morphology.},
  subject      = {Drosophila},
  language  = {en}
}
@phdthesis{Kunz2021,
  author    = {Kunz, Tobias C.},
  title     = {Expansion Microscopy (ExM) as a tool to study organelles and intracellular pathogens},
  doi       = {10.25972/OPUS-22333},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-223330},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {The resolution of fluorescence light microscopy was long believed to be limited by the diffraction limit of light of around 200-250 nm described in 1873 by Ernst Abbe. Within the last decade, several approaches, such as structured illumination microscopy (SIM), stimulated emission depletion STED and (direct) stochastic optical reconstruction microscopy (d)STORM have been established to bypass the diffraction limit. However, such super-resolution techniques enabling a resolution <100 nm require specialized and expensive setups as well as expert knowledge in order to avoid artifacts. They are therefore limited to specialized laboratories. Recently, Boyden and colleagues introduced an alternate approach, termed expansion microscopy (ExM). The latter offers the possibility to perform superresolution microscopy on conventional confocal microscopes by embedding the sample into a swellable hydrogel that is isotropically expanded. Since its introduction in 2015, expansion microscopy has developed rapidly offering protocols for 4x, 10x and 20x expansion of proteins and RNA in cells, tissues and human clinical specimens. Mitochondria are double membrane-bound organelles and crucial to the cell by performing numerous tasks, from ATP production through oxidative phosphorylation, production of many important metabolites, cell signaling to the regulation of apoptosis. The inner mitochondrial membrane is strongly folded forming so-called cristae. Besides being the location of the oxidative phosphorylation and therefore energy conversion and ATP production, cristae have been of great interest because changes in morphology have been linked to a plethora of diseases from cancer, diabetes, neurodegenerative diseases, to aging and infection. However, cristae imaging remains challenging as the distance between two individual cristae is often below 100 nm. Within this work, we demonstrate that the mitochondrial creatine kinase MtCK linked to fluorescent protein GFP (MtCK-GFP) can be used as a cristae marker. Upon fourfold expansion, we illustrate that our novel marker enables visualization of cristae morphology and localization of mitochondrial proteins relative to cristae without the need for specialized setups. Furthermore, we show the applicability of expansion microscopy for several bacterial pathogens, such as Chlamydia trachomatis, Simkania negevensis, Neisseria gonorrhoeae and Staphylococcus aureus. Due to differences in bacterial cell walls, we reveal important aspects for the digestion of pathogens for isotropic expansion. We further show that expansion of the intracellular pathogens C. trachomatis and S. negevensis, enables the differentiation between the two distinct developmental forms, catabolic active reticulate bodies (RB) and infectious elementary bodies (EB), on a conventional confocal microscope. We demonstrate the possibility to precisely locate chlamydial effector proteins, such as CPAF or Cdu1, within and outside the chlamydial inclusion. Moreover, we show that expansion microscopy enables the investigation of bacteria, herein S. aureus, within LAMP1 and LC3-II vesicles. With the introduction of the unnatural α-NH2-ω-N3-C6-ceramide, we further present the first approach for the expansion of lipids that may also be suitable for far inaccessible molecule classes like carbohydrates. The efficient accumulation and high labeling density of our functionalized α-NH2-ω-N3-C6-ceramide in both cells and bacteria enables in combination with tenfold expansion nanoscale resolution (10-20 nm) of the interaction of proteins with the plasma membrane, membrane of organelles and bacteria. Ceramide is the central molecule of the sphingolipid metabolism, an important constituent of cellular membranes and regulates many important cellular processes such as differentiation, proliferation and apoptosis. Many studies report about the importance of sphingolipids during infection of various pathogens. While the transport of ceramide to Chlamydia has been reported earlier, one of the unanswered questions remaining was if ceramide forms parts of the outer or inner bacterial membrane. Expansion of α-NH2-ω-N3-C6-ceramide enabled the visualization of ceramide in the inner and outer membrane of C. trachomatis and their distance was determined to be 27.6 ± 7.7 nm.},
  subject      = {Fluoreszenzmikroskopie},
  language  = {en}
}
@phdthesis{Zwettler2021,
  author    = {Zwettler, Fabian Ulrich},
  title     = {Expansionsmikroskopie kombiniert mit hochaufl{\"o}sender Fluoreszenzmikroskopie},
  doi       = {10.25972/OPUS-21236},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-212362},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Fluorescence microscopy is a form of light microscopy that has developed during the 20th century and is nowadays a standard tool in Molecular and Cell biology for studying the structure and function of biological molecules. High-resolution fluorescence microscopy techniques, such as dSTORM (direct Stochastic Optical Reconstruction Microscopy) allow the visualization of cellular structures at the nanometre scale (10-9 m). This has already made it possible to decipher the composition and function of various biopolymers, such as proteins, lipids and nucleic acids, up to the three-dimensional (3D) structure of entire organelles. In practice, however, it has been shown that these imaging methods and their further developments still face great challenges in order to achieve an effective resolution below ∼ 10 nm. This is mainly due to the nature of labelling biomolecules. For the detection of molecular structures, immunostaining is often performed as a standard method. Antibodies to which fluorescent molecules are coupled, recognize and bind specifcally and with high affnity to the molecular section of the target structure, also called epitope or antigen. The fluorescent molecules serve as reporter molecules which are imaged with the use of a fluorescence microscope. However, the size of these labels with a length of about 10-15 nm in the case of immunoglobulin G (IgG) antibodies, cause a detection of the fluorescent molecules shifted to the real position of the studied antigen. In dense regions where epitopes are located close to each other, steric hindrance between antibodies can also occur and leads to an insuffcient label density. Together with the shifted detection of fluorescent molecules, these factors can limit the achievable resolution of a microscopy technique. Expansion microscopy (ExM) is a recently developed technique that achieves a resolution improvement by physical expansion of an investigated object. Therefore, biological samples such as cultured cells, tissue sections, whole organs or isolated organelles are chemically anchored into a swellable polymer. By absorbing water, this so-called superabsorber increases its own volume and pulls the covalently bound biomolecules isotropically apart. Routinely, this method achieves a magnifcation of the sample by about four times its volume. But protocol variants have already been developed that result in higher expansion factors of up to 50-fold. Since the ExM technique includes in the frst instance only the sample treatment for anchoring and magnifcation of the sample, it can be combined with various standard methods of fluorescence microscopy. In theory, the resolution of the used imaging technique improves linearly with the expansion factor of the ExM treated sample. However, an insuffcient label density and the size of the antibodies can here again impair the effective achievable resolution. The combination of ExM with high-resolution fluorescence microscopy methods represents a promising strategy to increase the resolution of light microscopy. In this thesis, I will present several ExM variants I developed which show the combination of ExM with confocal microscopy, SIM (Structured Illumination Microscopy), STED (STimulated Emission Depletion) and dSTORM. I optimized existing ExM protocols and developed different expansion strategies, which allow the combination with the respective imaging technique. Thereby, I gained new structural insights of isolated centrioles from the green algae Chlamydomonas reinhardtii by combining ExM with STED and confocal microscopy. In another project, I combined 3D-SIM imaging with ExM and investigated the molecular structure of the so-called synaptonemal complex. This structure is formed during meiosis in eukaryotic cells and contributes to the exchange of genetic material between homologous chromosomes. Especially in combination with dSTORM, the ExM method showed its high potential to overcome the limitations of modern fluorescence microscopy techniques. In this project, I expanded microtubules in mammalian cells, a polymer of the cytoskeleton as well as isolated centrioles from C. reinhardtii. By labelling after expansion of the samples, I was able to signifcantly reduce the linkage error of the label and achieve an improved label density. In future, these advantages together with the single molecule sensitivity and high resolution obtained by the dSTORM method could pave the way for achieving molecular resolution in fluorescence microscopy},
  subject      = {Fluoreszenzmikroskopie},
  language  = {en}
}
@phdthesis{Lehmann2020,
  author    = {Lehmann, Julian},
  title     = {Hochaufl{\"o}sende Fluoreszenzmikroskopie beleuchtet den Oligomerisierungsstatus pflanzlicher Membranproteine},
  doi       = {10.25972/OPUS-21176},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-211762},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {SLAC/SLAH Anionenkan{\"a}le, die zur Familie der langsamen Anionenkan{\"a}le geh{\"o}ren, repr{\"a}sentieren Schl{\"u}sselproteine in der pflanzlichen Stressantwort. Neben ihrer Aufgabe in Stresssituationen, ist eine Untergruppe der Kan{\"a}le f{\"u}r die Beladung der Leitgef{\"a}ße mit Nitrat und Chlorid in der Stele der Pflanzenwurzeln verantwortlich. Biophysikalische und pflanzenphysiologische Studien stellten heraus, dass vor Allem der Anionenkanal SLAH3 f{\"u}r die Beladung der Xylem Leitgef{\"a}ße mit Nitrat und Chlorid verantwortlich ist. Ihm zur Seite gestellt werden noch die elektrisch inaktiven Homologe SLAH1 und SLAH4 in der Wurzel exprimiert. Sie steuern die Aktivit{\"a}t von SLAH3 durch die Assemblierung zu SLAH1/SLAH3 oder SLAH3/SLAH4 Heteromeren. Neben der Kontrolle durch Heteromerisierungsereignisse, werden SLAH3 Homomere sehr spezifisch und schnell durch zytosolische Ans{\"a}uerung aktiviert. Obwohl bereits die Kristallstruktur des bakteriellen Homologs HiTehA zu pflanzlichen SLAC/SLAH Anionenkan{\"a}len bekannt ist, welche HiTehA als Trimer charakterisiert, sind die St{\"o}chiometrie und der Polymerisierungsgrad der pflanzlichen SLAC/SLAHs bisher noch unbekannt. Die Fluoreszenzmikroskopie umfasst viele etablierte Anwendungsmethoden, wie die konfokale Laserrastermikroskopie (CLSM), Techniken mit verbesserter Aufl{\"o}sung, wie die Mikroskopie mit strukturierter Beleuchtung (SIM) und hochaufl{\"o}sende Methoden, welche durch die Lokalisationsmikroskopie (z.B. dSTORM und PALM) oder die Expansionsmikroskopie (ExM) vertreten werden. Diese unterschiedlichen Mikroskopie-methoden erm{\"o}glichen neue Einblicke in die Organisation von Proteinen in biologischen Systemen, die bis auf die molekulare Ebene hinunterreichen. Insbesondere im Bereich der hochaufl{\"o}senden Fluoreszenzmikroskopie sind im Gegensatz zu tierischen Frage-stellungen bisher jedoch nur wenige Untersuchungen in pflanzlichen Geweben durchgef{\"u}hrt worden. Die Lokalisationsmikroskopie erm{\"o}glicht die Quantifizierung einzelner Molek{\"u}le in nativen Systemen und l{\"a}sst {\"u}berdies R{\"u}ckschl{\"u}sse auf den Polymerisierungsgrad von Proteinen zu. Da Poly- und Heteromerisierung von Proteinen oftmals mit der Funktionalit{\"a}t eines entsprechenden Proteins einhergeht, wie es bei den SLAC/SLAH Anionenkan{\"a}len der Fall ist, wurden in dieser Arbeit PALM Messungen zur Untersuchung des Polymerisierungsgrades und Interaktionsmuster der Anionenkan{\"a}le angewendet. Ferner wurden Expressionsmuster der SLAC/SLAHs untersucht und zudem Mikroskopieanwendungen im Pflanzengewebe etabliert und verbessert. In Bezug auf die Mikroskopieanwendungen konnten wir in Arabidopsis thaliana (At) Wurzeln die polare Verteilung von PIN Proteinen mittels SIM best{\"a}tigen und die gruppierte Verteilung in der Plasmamembran am Zellpol aufl{\"o}sen. In Wurzel-querschnitten war es m{\"o}glich, Zellw{\"a}nde zu vermessen, den Aufbau der Pflanzenwurzel mit den verschiedenen Zelltypen zu rekonstruieren und diesen in Zusammenhang mit Zellwanddicken zu bringen. Anhand dieser Aufnahmen ließ sich die Aufl{\"o}sungsgrenze eines SIM-Mikroskops bestimmen, weshalb diese Probe als Modellstruktur f{\"u}r Aufl{\"o}sungsanalysen, zur Kontrolle f{\"u}r die korrekte Bildverarbeitung bei hochaufl{\"o}sender Bildgebung und andere Fragestellungen empfohlen werden kann. F{\"u}r die Expansionsmikroskopie in pflanzlichen Proben konnten ein enzym- und ein denaturierungsbasiertes Pr{\"a}parationsprotokoll etabliert werden. Dabei wurden ganze At Setzlinge, Wurzelabschnitte und Blattst{\"u}cke gef{\"a}rbt, expandiert und mit zwei bis drei Mal verbesserter Aufl{\"o}sung bildlich dargestellt. In diesem Zusammenhang waren Aufnahmen ganzer Wurzel- und Blattproben mit beeindruckender Eindringtiefe und extrem geringem Hintergrundsignal m{\"o}glich. Zudem wurden die Daten kritisch betrachtet, Probleme aufgezeigt, gewebespezifische Ver{\"a}nderungen dargestellt und limitierende Faktoren f{\"u}r die ExM in Pflanzenproben thematisiert. Im Fokus dieser Arbeit stand die Untersuchung der SLAC/SLAH Proteine. SLAH2 wird in den Wurzeln vornehmlich in Endodermis- und Perizykelzellen exprimiert, was anhand verschiedener At SLAH2 YFP Mutanten untersucht werden konnte. Dies unterst{\"u}tzt die Annahme, dass SLAH2 bei der Beladung der Leitgef{\"a}ße mit Nitrat maßgeblich beteiligt ist. Es ist denkbar, dass SLAH2 ebenfalls eine wachstumsbeeinflussende Funktion {\"u}ber die Regulation von Nitratkonzentrationen zugeschrieben werden kann. Darauf deuten vor allem die verst{\"a}rkte Expression von SLAH2 im Bereich der Seitenwurzeln und die heterogene Expression in der Elongations-, Differenzierungs- und meristematischen Zone hin. Die Membranst{\"a}ndigkeit von SLAH4 konnte nachgewiesen werden und FRET FLIM Untersuchungen zeigten eine hohe Affinit{\"a}t von SLAH4 zu SLAH3, was die beiden Homologe als Interaktionspartner identifiziert. F{\"u}r die Bestimmung des Oligomerisierungsgrades mittels PALM wurden die pflanzlichen Anionenkan{\"a}le in tierischen COS7-Zellen exprimiert. Die elektrophysiologische Funktionalit{\"a}t der mEOS2-SLAC/SLAH-Konstrukte wurde mit Hilfe von Patch-Clamp-Versuchen in COS7-Zellen {\"u}berpr{\"u}ft. Um Expressionslevel, Membranst{\"a}ndigkeit und die Verteilung {\"u}ber die Membran der SLAC/SLAHs zu verifizieren, wurden dSTORM-Aufnahmen herangezogen Schließlich erm{\"o}glichten PALM-Aufnahmen die Bestimmung des Polymerisierungs-grades der SLAC/SLAH Anionenkan{\"a}le, die st{\"o}chiometrischen Ver{\"a}nderungen bei Heteromerisierung von SLAH3 mit SLAH1 oder SLAH4 und auch der Einfluss einer zytosolischer Ans{\"a}uerung auf den Polymerisierungsgrad von SLAH3 Homomeren. Zudem weisen die Oligomerisierungsanalysen von SLAH3 Mutanten darauf hin, dass die Aminos{\"a}uren Histidin His330 und His454 entscheidend an der pH sensitiven Regulierung von SLAH3 beteiligt sind. Durch die erhobenen Daten konnten also entscheidende, neue Erkenntnisse {\"u}ber die Regulationsmechanismen von pflanzlichen Anionenkan{\"a}len auf molekularer Ebene gewonnen werden: Unter Standardbedingungen liegen SLAC1, SLAH2 und SLAH3 haupts{\"a}chlich als Dimer vor. Auf eine zytosolische Ans{\"a}uerung reagiert ausschließlich SLAH3 mit einer signifikanten st{\"o}chiometrischen Ver{\"a}nderung und liegt im aktiven Zustand vor Allem als Monomer vor. Der Oligomerisierungsgrad von SLAC1 und SLAH2 bleibt hingegen bei einer zytosolischen Ans{\"a}uerung unver{\"a}ndert. Ferner kommt es bei der Interaktion von SLAH3 mit SLAH1 oder SLAH4 zur Formierung eines Heterodimers, welches unbeeinflusst durch den zytosolischen pH bleibt. Im Gegensatz dazu bleiben die elektrisch inaktiven Untereinheiten SLAH1 und SLAH4 monomerisch und assemblieren ganz spezifisch nur mit SLAH3. Die hochaufl{\"o}sende Fluoreszenz-mikroskopie, insbesondere PALM erlaubt es also Heteromerisierungsereignisse und {\"A}nderungen im Poylmerisierungsgrad von Membranproteinen wie den SLAC/SLAHs auf molekularer Ebene zu untersuchen und l{\"a}sst so R{\"u}ckschl{\"u}sse auf physiologische Ereignisse zu.},
  subject      = {Fluoreszenzmikroskopie},
  language  = {de}
}
@phdthesis{Heil2020,
  author    = {Heil, Hannah Sophie},
  title     = {Sharpening super-resolution by single molecule localization microscopy in front of a tuned mirror},
  doi       = {10.25972/OPUS-20432},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-204329},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {The „Resolution Revolution" in fluorescence microscopy over the last decade has given rise to a variety of techniques that allow imaging beyond the diffraction limit with a resolution power down into the nanometer range. With this, the field of so-called super-resolution microscopy was born. It allows to visualize cellular architecture at a molecular level and thereby achieve a resolution level that had been previously only accessible by electron microscopy approaches. One of these promising techniques is single molecule localization microscopy (SMLM) in its most varied forms such as direct stochastic optical reconstruction microscopy (dSTORM) which are based on the temporal separation of the emission of individual fluorophores. Localization analysis of the subsequently taken images of single emitters eventually allows to reconstruct an image containing super-resolution information down to typically 20 nm in a cellular setting. The key point here is the localization precision, which mainly depends on the image contrast generated the by the individual fluorophore's emission. Thus, measures to enhance the signal intensity or reduce the signal background allow to increase the image resolution achieved by dSTORM. In my thesis, this is achieved by simply adding a reflective metal-dielectric nano-coating to the microscopy coverslip that serves as a tunable nano-mirror. I have demonstrated that such metal-dielectric coatings provide higher photon yield at lower background and thus substantially improve SMLM performance by a significantly increased localization precision, and thus ultimately higher image resolution. The strength of this approach is that ─ except for the coated cover glass ─ no specialized setup is required. The biocompatible metal-dielectric nano-coatings are fabricated directly on microscopy coverslips and have a simple three-ply design permitting straightforward implementation into a conventional fluorescence microscope. The introduced improved lateral resolution with such mirror-enhanced STORM (meSTORM) not only allows to exceed Widefield and Total Internal Reflection Fluorescence (TIRF) dSTORM performance, but also offers the possibility to measure in a simplified setup as it does not require a special TIRF objective lens. The resolution improvement achieved with meSTORM is both spectrally and spatially tunable and thus allows for dual-color approaches on the one hand, and selectively highlighting region above the cover glass on the other hand, as demonstrated here. Beyond lateral resolution enhancement, the clear-cut profile of the highlighted region provides additional access to the axial dimension. As shown in my thesis, this allows for example to assess the three-dimensional architecture of the intracellular microtubule network by translating the local localization uncertainty to a relative axial position. Even beyond meSTORM, a wide range of membrane or surface imaging applications may benefit from the selective highlighting and fluorescence enhancing provided by the metal-dielectric nano-coatings. This includes for example, among others, live-cell Fluorescence Correlation Spectroscopy and Fluorescence Resonance Energy Transfer studies as recently demonstrated.},
  subject      = {Fluoreszenz},
  language  = {en}
}
@phdthesis{Gorelashvili2019,
  author    = {Gorelashvili, Maximilian Georg},
  title     = {Investigation of megakaryopoiesis and the acute phase of ischemic stroke by advanced fluorescence microscopy},
  doi       = {10.25972/OPUS-18600},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-186002},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {In mammals, anucleate platelets circulate in the blood flow and are primarily responsible for maintaining functional hemostasis. Platelets are generated in the bone marrow (BM) by megakaryocytes (MKs), which mainly reside directly next to the BM sinusoids to release proplatelets into the blood. MKs originate from hematopoietic stem cells and are thought to migrate from the endosteal to the vascular niche during their maturation, a process, which is, despite being intensively investigated, still not fully understood. Long-term intravital two photon microscopy (2PM) of MKs and vasculature in murine bone marrow was performed and mean squared displacement analysis of cell migration was performed. The MKs exhibited no migration, but wobbling-like movement on time scales of 3 h. Directed cell migration always results in non-random spatial distribution. Thus, a computational modelling algorithm simulating random MK distribution using real 3D light-sheet fluorescence microscopy data sets was developed. Direct comparison of real and simulated random MK distributions showed, that MKs exhibit a strong bias to vessel-contact. However, this bias is not caused by cell migration, as non-vessel-associated MKs were randomly distributed in the intervascular space. Furthermore, simulation studies revealed that MKs strongly impair migration of other cells in the bone marrow by acting as large-sized obstacles. MKs are thought to migrate from the regions close to the endosteum towards the vasculature during their maturation process. MK distribution as a function of their localization relative to the endosteal regions of the bones was investigated by light sheet fluorescence microscopy (LSFM). The results show no bone-region dependent distribution of MKs. Taken together, the newly established methods and obtained results refute the model of MK migration during their maturation. Ischemia reperfusion (I/R) injury is a frequent complication of cerebral ischemic stroke, where brain tissue damage occurs despite successful recanalization. Platelets, endothelial cells and immune cells have been demonstrated to affect the progression of I/R injury in experimental mouse models 24 h after recanalization. However, the underlying Pathomechanisms, especially in the first hours after recanalization, are poorly understood. Here, LSFM, 2PM and complemental advanced image analysis workflows were established for investigation of platelets, the vasculature and neutrophils in ischemic brains. Quantitative analysis of thrombus formation in the ipsilateral and contralateral hemispheres at different time points revealed that platelet aggregate formation is minimal during the first 8 h after recanalization and occurs in both hemispheres. Considering that maximal tissue damage already is present at this time point, it can be concluded that infarct progression and neurological damage do not result from platelet aggregated formation. Furthermore, LSFM allowed to confirm neutrophil infiltration into the infarcted hemisphere and, here, the levels of endothelial cell marker PECAM1 were strongly reduced. However, further investigations must be carried out to clearly identify the role of neutrophils and the endothelial cells in I/R injury.},
  subject      = {Fluoreszenzmikroskopie},
  language  = {en}
}
@phdthesis{Letschert2019,
  author    = {Letschert, Sebastian},
  title     = {Quantitative Analysis of Membrane Components using Super-Resolution Microscopy},
  doi       = {10.25972/OPUS-16213},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-162139},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {The plasma membrane is one of the most thoroughly studied and at the same time most complex, diverse, and least understood cellular structures. Its function is determined by the molecular composition as well as the spatial arrangement of its components. Even after decades of extensive membrane research and the proposal of dozens of models and theories, the structural organization of plasma membranes remains largely unknown. Modern imaging tools such as super-resolution fluorescence microscopy are one of the most efficient techniques in life sciences and are widely used to study the spatial arrangement and quantitative behavior of biomolecules in fixed and living cells. In this work, direct stochastic optical reconstruction microscopy (dSTORM) was used to investigate the structural distribution of mem-brane components with virtually molecular resolution. Key issues are different preparation and staining strategies for membrane imaging as well as localization-based quantitative analyses of membrane molecules. An essential precondition for the spatial and quantitative analysis of membrane components is the prevention of photoswitching artifacts in reconstructed localization microscopy images. Therefore, the impact of irradiation intensity, label density and photoswitching behavior on the distribution of plasma membrane and mitochondrial membrane proteins in dSTORM images was investigated. It is demonstrated that the combination of densely labeled plasma membranes and inappropriate photoswitching rates induces artificial membrane clusters. Moreover, inhomogeneous localization distributions induced by projections of three-dimensional membrane structures such as microvilli and vesicles are prone to generate artifacts in images of biological membranes. Alternative imaging techniques and ways to prevent artifacts in single-molecule localization microscopy are presented and extensively discussed. Another central topic addresses the spatial organization of glycosylated components covering the cell membrane. It is shown that a bioorthogonal chemical reporter system consisting of modified monosaccharide precursors and organic fluorophores can be used for specific labeling of membrane-associated glycoproteins and -lipids. The distribution of glycans was visualized by dSTORM showing a homogeneous molecule distribution on different mammalian cell lines without the presence of clusters. An absolute number of around five million glycans per cell was estimated and the results show that the combination of metabolic labeling, click chemistry, and single-molecule localization microscopy can be efficiently used to study cell surface glycoconjugates. In a third project, dSTORM was performed to investigate low-expressing receptors on cancer cells which can act as targets in personalized immunotherapy. Primary multiple myeloma cells derived from the bone marrow of several patients were analyzed for CD19 expression as potential target for chimeric antigen receptor (CAR)-modified T cells. Depending on the patient, 60-1,600 CD19 molecules per cell were quantified and functional in vitro tests demonstrate that the threshold for CD19 CAR T recognition is below 100 CD19 molecules per target cell. Results are compared with flow cytometry data, and the important roles of efficient labeling and appropriate control experiments are discussed.},
  subject      = {Fluoreszenzmikroskopie},
  language  = {en}
}
@phdthesis{Schmithausen2019,
  author    = {Schmithausen, Patrick Alexander Gerhard},
  title     = {Three-dimensional fluorescence image analysis of megakaryocytes and vascular structures in intact bone},
  doi       = {10.25972/OPUS-17854},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-178541},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {The thesis provides insights in reconstruction and analysis pipelines for processing of three-dimensional cell and vessel images of megakaryopoiesis in intact murine bone. The images were captured in a Light Sheet Fluorescence Microscope. The work presented here is part of Collaborative Research Centre (CRC) 688 (project B07) of the University of W{\"u}rzburg, performed at the Rudolf-Virchow Center. Despite ongoing research within the field of megakaryopoiesis, its spatio-temporal pattern of megakaryopoiesis is largely unknown. Deeper insight to this field is highly desirable to promote development of new therapeutic strategies for conditions related to thrombocytopathy as well as thrombocytopenia. The current concept of megakaryopoiesis is largely based on data from cryosectioning or in vitro studies indicating the existence of spatial niches within the bone marrow where specific stages of megakaryopoiesis take place. Since classic imaging of bone sections is typically limited to selective two-dimensional views and prone to cutting artefacts, imaging of intact murine bone is highly desired. However, this has its own challenges to meet, particularly in image reconstruction. Here, I worked on processing pipelines to account for irregular specimen staining or attenuation as well as the extreme heterogeneity of megakaryocyte morphology. Specific challenges for imaging and image reconstruction are tackled and solution strategies as well as remaining limitations are presented and discussed. Fortunately, modern image processing and segmentation strongly benefits from continuous advances in hardware as well as software-development. This thesis exemplifies how a combined effort in biomedicine, computer vision, data processing and image technology leads to deeper understanding of megakaryopoiesis. Tailored imaging pipelines significantly helped elucidating that the large megakaryocytes are broadly distributed throughout the bone marrow facing a surprisingly dense vessel network. No evidence was found for spatial niches in the bone marrow, eventually resulting in a revised model of megakaryopoiesis.},
  subject      = {Megakaryozytopoese},
  language  = {en}
}
@phdthesis{Schreiber2018,
  author    = {Schreiber, Benjamin},
  title     = {Selective and enhanced fluorescence by biocompatible nanocoatings to monitor G-protein-coupled receptor dynamics},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-173923},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Fluorescence microscopy has become one of the most important techniques for the imaging of biological cells and tissue, since the technique allows for selective labeling with fluorescent molecules and is highly suitable for low-light applications down to the single molecule regime. The methodological requirements are well-defined for studying membrane receptors within a highly localized nanometer-thin membrane. For example, G-protein-coupled receptors (GPCRs) are an extensively studied class of membrane receptors that represent one of the most important pharmaceutical targets. Ligand binding and GPCR activation dynamics are suspected to take place at the millisecond scale and may even be far faster. Thus, techniques that are fast, selective, and live-cell compatible are required to monitor GPCR dynamics. Fluorescence resonance energy transfer (FRET) and total internal reflection fluorescence microscopy (TIRF-M) are methods of choice to monitor the dynamics of GPCRs selectively within the cell membrane. Despite the remarkable success of these modalities, there are limitations. Most importantly, inhomogeneous illumination can induce imaging artifacts, rendering spectroscopic evaluation difficult. Background signal due to scattering processes or imperfect labeling can hamper the signal-to-noise, thus limiting image contrast and acquisition speed. Careful consideration of the internal physiology is required for FRET sensor design, so that ligand binding and cell compatibility are well-preserved despite the fluorescence labeling procedures. This limitation of labeling positions leads to very low signal changes in FRET-based GPCR analysis. In addition, microscopy of these systems becomes even more challenging in single molecule or low-light applications where the accuracy and temporal resolution may become dramatically low. Fluorescent labels should therefore be brighter, protected from photobleaching, and as small as possible to avoid interference with the binding kinetics. The development of new fluorescent molecules and labeling methods is an ongoing process. However, a complete characterization of new labels and sensors takes time. So far, the perfect dye system for GPCR studies has not been found, even though there is high demand. Thus, this thesis explores and applies a different approach based on improved illumination schemes for TIRF-M as well as metal-coated coverslips to enhance fluorescence and FRET efficiency. First, it is demonstrated that a 360° illumination scheme reduces typical TIRF artifacts and produces a much more homogenously illuminated field of view. Second, membrane imaging and FRET spectroscopy are improved by metal coatings that are used to modulate the fluorescent properties of common fluorescent dyes. Computer simulation methods are used to understand the underlying photophysics and to design the coatings. Third, this thesis explores the operational regime and limitations of plasmonic approaches with high sectioning capabilities. The findings are summarized by three publications that are presented in the results section of this work. In addition, the theory of fluorescence and FRET is explained, with particular attention to its emission modulations in the vicinity of metal-dielectric layers. Details of the instrumentation, computer simulations, and cell culture are described in the method section. The work concludes with a discussion of the findings within the framework of recent technological developments as well as perspectives and suggestions for future approaches complete the presented work.},
  subject      = {G-Protein gekoppelte Rezeptoren},
  language  = {en}
}
@phdthesis{Glogger2018,
  author    = {Glogger, Marius},
  title     = {Single-molecule fluorescence microscopy in live \(Trypanosoma\) \(brucei\) and model membranes},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-169222},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Der eukaryotische Parasit Trypanosoma brucei hat komplexe Strategien entwickelt um der Immunantwort eines Wirtes zu entkommen und eine persistente Infektion innerhalb dessen aufrechtzuerhalten. Ein zentrales Element seiner Verteidigungsstrategie st{\"u}tzt sich auf die Schutzfunktion seines Proteinmantels auf der Zelloberfl{\"a}che. Dieser Mantel besteht aus einer dichten Schicht aus identischen, Glykosylphosphatidylinositol (GPI)-verankerten variablen Oberfl{\"a}chenglykoproteinen (VSG). Der VSG Mantel verhindert die Erkennung der darunterliegenden, invarianten Epitope durch das Immunsystem. Obwohl es notwendig ist die Funktionsweise des VSG Mantels zu verstehen, vor allem um ihn als m{\"o}gliches Angriffsziel gegen den Parasiten zu verwenden, sind seine biophysikalischen Eigenschaften bisher nur unzureichend verstanden. Dies ist vor allem der Tatsache geschuldet, dass die hohe Motilit{\"a}t der Parasiten mikroskopische Studien in lebenden Zellen bisher weitestgehend verhinderten. In der vorliegenden Arbeit wird nun hochmoderne Einzelmolek{\"u}l-Fluoreszenzmikroskopie (EMFM) als M{\"o}glichkeit f{\"u}r mikroskopische Untersuchungen im Forschungsbereich der Trypanosomen vorgestellt. Die Arbeit umfasst Untersuchungen der VSG Dynamik unter definierten Bedingungen k{\"u}nstlicher Membransysteme. Es wurde zuerst der Einfluss der lateralen Proteindichte auf die VSG Diffusion untersucht. Experimente mittels Fluoreszenz- Wiederkehr nach irreversiblem Photobleichen und komplement{\"a}re Einzelmolek{\"u}l- Verfolgungs Experimente offenbarten, dass ein molekularer Diffusionsschwellenwert existiert. {\"U}ber diesem Schwellenwert wurde eine dichteabh{\"a}nige Reduzierung des Diffusionskoeffizienten gemessen. Eine relative Quantifizierung der rekonstituierten VSGs verdeutlichte, dass der Oberfl{\"a}chenmantel der Trypanosomen sehr nahe an diesem Schwellenwert agiert. Der VSG Mantel ist optimiert um eine hohe Proteindichte bei gleichzeitiger hoher Mobilit{\"a}t der VSGs zu gew{\"a}hrleisten. Des Weiteren wurde der Einfluss der VSG N-Glykosylierung auf die Diffusion des Proteins quantitativ untersucht. Die Messungen ergaben, dass die N-Glykosylierung dazu beitr{\"a}gt eine hohe Mobilit{\"a}t bei hohen Proteindichten aufrechtzuerhalten. Eine detaillierte Analyse von VSG Trajektorien offenbarte, dass zwei unterschiedliche Populationen frei diffundierender VSGs in der k{\"u}nstlichen Membran vorlagen. K{\"u}rzlich wurde entdeckt, dass VSGs zwei strukturell unterschiedliche Konformationen annehmen k{\"o}nnen. Die Messungen in der Arbeit stimmen mit diesen Beschreibungen {\"u}berein. Die Ergebnisse der EMFM in k{\"u}nstlichen Membranen wurden durch VSG Einzelmolek{\"u}l- Verfolgungs Experimente auf lebenden Zellen erg{\"a}nzt. Es wurde eine hohe Mobilit{\"a}t und Dynamik einzelner VSGs gemessen, was die allgemein dynamische Natur des VSG Mantels verdeutlicht. Dies f{\"u}hrte zu der Schlussfolgerung, dass der VSG Mantel auf lebenden Trypanosomen ein dichter und dennoch dynamischer Schutzmantel ist. Die F{\"a}higkeit der VSGs ihre Konformation flexibel anzupassen, unterst{\"u}tzt das Erhalten der Fluidit{\"a}t bei variablen Dichten. Diese Eigenschaften des VSG Mantels sind elementar f{\"u}r die Aufrechterhaltung einer presistenden Infektion eines Wirtes. In dieser Arbeit werden des Weiteren verschiedene, auf Hydrogel basierende Einbettungsmethoden vorgestellt. Diese erm{\"o}glichten die Zellimmobilisierung und erlaubten EMFM in lebenden Trypanosomen. Die Hydrogele wiesen eine hohe Zytokompatibilit{\"a}t auf. Die Zellen {\"u}berlebten in den Gelen f{\"u}r eine Stunde nach Beginn der Immobilisierung. Die Hydrogele erf{\"u}llten die Anforderungen der Superresolution Mikroskopie (SRM) da sie eine geringe Autofluoreszenz im Spektralbereich der verwendeten Fluorophore besaßen. Mittels SRM konnte nachgewiesen werden, dass die Hydrogele die Zellen effizient immobilisierten. Als erstes Anwendungsbeispiel der Methode wurde die Organisation der Plasmamembran in lebenden Trypanosomen untersucht. Die Untersuchung eines fluoreszenten Tracers in der inneren Membranschicht ergab, dass dessen Verteilung nicht homogen war. Es wurden spezifische Membrandom{\"a}nen gefunden, in denen das Molek{\"u}l entweder vermehrt oder vermindert auftrat. Dies f{\"u}hrte zu der Schlussfolgerung, dass diese Verteilung durch eine Interaktion des Tracers mit Proteinen des zellul{\"a}ren Zytoskeletts zustande kam. Die in dieser Arbeit pr{\"a}sentierten Ergebnisse zeigen, dass EMFM erfolgreich f{\"u}r verschiedene biologische Untersuchungen im Forschungsfeld der Trypanosomen angewendet werden kann. Dies gilt zum Beispiel f{\"u}r die Untersuchung von der VSG Dynamik in k{\"u}nstlichen Membransystemen, aber auch f{\"u}r Studien in lebenden Zellen unter Verwendung der auf Hydrogelen basierenden Zelleinbettung.},
  subject      = {Trypanosoma brucei},
  language  = {en}
}
@phdthesis{Bartossek2018,
  author    = {Bartossek, Thomas},
  title     = {Structural and functional analysis of the trypanosomal variant surface glycoprotein using x-ray scattering techniques and fluorescence microscopy},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-144775},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Trypanosoma brucei is an obligate parasite and causative agent of severe diseases affecting humans and livestock. The protist lives extracellularly in the bloodstream of the mammalian host, where it is prone to attacks by the host immune system. As a sophisticated means of defence against the immune response, the parasite's surface is coated in a dense layer of the variant surface glycoprotein (VSG), that reduces identification of invariant epitopes on the cell surface by the immune system to levels that prevent host immunity. The VSG has to form a coat that is both dense and mobile, to shield invariant surface proteins from detection and to allow quick recycling of the protective coat during immune evasion. This coat effectively protects the parasite from the harsh environment that is the mammalian bloodstream and leads to a persistent parasitemia if the infection remains untreated. The available treatment against African Trypanosomiasis involves the use of drugs that are themselves severely toxic and that can lead to the death of the patient. Most of the drugs used as treatment were developed in the early-to-mid 20th century, and while developments continue, they still represent the best medical means to fight the parasite. The discovery of a fluorescent VSG gave rise to speculations about a potential interaction between the VSG coat and components of the surrounding medium, that could also lead to a new approach in the treatment of African Trypanosomiasis that involves the VSG coat. The initially observed fluorescence signal was specific for a combination of a VSG called VSG'Y' and the triphenylmethane (TPM) dye phenol red. Exchanging this TPM to a bromo-derivative led to the observation of another fluorescence effect termed trypanicidal effect which killed the parasite independent of the expressed VSG and suggests a structurally conserved feature between VSGs that could function as a specific drug target against T. b. brucei. The work of this thesis aims to identify the mechanisms that govern the unique VSG'Y' fluorescence and the trypanocidal effect. Fluorescence experiments and protein mutagenesis of VSG'Y' as well as crystallographic trials with a range of different VSGs were utilized in the endeavour to identify the binding mechanisms between TPM compounds and VSGs, to find potentially conserved structural features between VSGs and to identify the working mechanisms of VSG fluorescence and the trypanocidal effect. These trials have the potential to lead to the formulation of highly specific drugs that target the parasites VSG coat. During the crystallographic trials of this thesis, the complete structure of a VSG was solved experimentally for the first time. This complete structure is a key component in furthering the understanding of the mechanisms governing VSG coat formation. X-ray scattering techniques, involving x-ray crystallography and small angle x-ray scattering were applied to elucidate the first complete VSG structures, which reveal high flexibility of the protein and supplies insight into the importance of this flexibility in the formation of a densely packed but highly mobile surface coat.},
  subject      = {Trypanosoma brucei brucei},
  language  = {en}
}
@phdthesis{Burgert2018,
  author    = {Burgert, Anne},
  title     = {Untersuchung von Sphingolipiden und anderen Membrankonjugaten mittels hochaufl{\"o}sender Fluoreszenzmikroskopie},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-145725},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Methoden der Fluoreszenz-Lokalisationsmikroskopie (engl. single-molecule localization microscopy, SMLM) erm{\"o}glichen es Molek{\"u}le zu quantifizieren und deren Verteilung zu analysieren. Im Rahmen dieser Arbeit wurden verschiedene Membranmolek{\"u}le auf unterschiedlichen eukaryotischen Zellen, aber auch auf Prokaryoten mit dSTORM (engl. direct stochastic optical reconstruction microscopy) oder PALM (engl.: photoactivated localization microscopy) aufgenommen und quantifiziert. Bevor jedoch diese hochaufl{\"o}sende fluoreszenzbasierte Technik f{\"u}r biologische Fragestellungen angewendet werden konnten, mussten zun{\"a}chst potentielle Artefakt-ausl{\"o}sende Quellen identifiziert und Strategien gefunden werden, um diese zu eliminieren. Eine m{\"o}gliche Artefakt-Quelle ist eine zu niedrige Photonenzahl, die von Fluorophoren emittiert wird. Werden zu wenige Photonen detektiert, kann die Lokalisation eines Fluorophors weniger pr{\"a}zise bestimmt werden. Dies kann zu einer falschen Abbildung von Strukturen f{\"u}hren oder zu falschen R{\"u}ckschl{\"u}ssen {\"u}ber die Verteilung von Molek{\"u}len. Eine M{\"o}glichkeit die Anzahl der emittierten Photonen zu erh{\"o}hen, ist chemische Additive als Triplettl{\"o}scher einzusetzen. Sie bewirken, dass die Fluorophore wieder in den Grundzustand relaxieren und somit wieder angeregt werden k{\"o}nnen. Es wurden verschiedene Additive, die in der Literatur als Triplettl{\"o}scher beschrieben sind, getestet. Dazu wurden zun{\"a}chst ihre Auswirkungen auf den Triplettzustand verschiedener Fluorophore (Alexa Fluor (Al) 488, 532 und 647 und Atto655) mit Hilfe von Fluoreszenzkorrelationsspektroskopie (FCS) untersucht. Cyclooctatetraen (COT) bewirkte dabei eine Abnahme der Triplettausbeute von Al488, Al532 und Al647 um ~ 40-60\%, bei Atto655 ver{\"a}nderte sie sich nicht. Obwohl die Ergebnisse der FCS-Messungen darauf hindeuten, dass COT in einer erh{\"o}hten Anzahl an emittierten Photonen resultiert, konnte dies bei dSTORM-Messungen nicht best{\"a}tigt werden. Hier hatte COT nur einen gr{\"o}ßeren positiven Effekt auf das Fluorophor Al647 (Zunahme um ~ 60\%). Eine Erkl{\"a}rung f{\"u}r diese Widerspr{\"u}chlichkeit zu den Ergebnissen aus den FCS-Messungen, k{\"o}nnte das Vorhandensein des Schaltpuffers bei dSTORM-Messungen sein. Dieser bewirkt den {\"U}bergang der Fluorophore in den Aus-Zustand bzw. entzieht dem Puffer Sauerstoff. Bei der Zugabe von 5 mM Kaliumiodid (KI) nahm die Triplettamplitude bei FCS-Messungen nur bei Al488 ab (um ~ 80\%). Eine geringe Steigerung (um ~ 10\%) der Intensit{\"a}t von Al488 mit KI konnte bei dSTORM-Messungen mit niedrigen Konzentrationen (~ 0,5 mM) erzielt werden. Bei einer Konzentration von 5 mM sank die Intensit{\"a}t jedoch wieder um 40\%. Deuteriumoxid (D2O) soll, anders als die Triplettl{\"o}scher, eine Verbesserung der Photonenausbeute dadurch bewirken, dass strahlungslose Relaxationsprozesse minimiert werden. Mit dSTORM-Messungen konnte gezeigt werden, dass Atto655 und Al647 in D2O zwar pro An-Zustand mehr Photonen emittieren als in Schaltpuffer ohne D2O, da die Fluorophore hier jedoch schneller bleichen, letztendlich die gleiche Anzahl an Photonen detektiert werden. Um die Anzahl an emittierten Photonen zu erh{\"o}hen, eignet sich also nur COT bei dSTORM-Messungen mit AL647 und KI in sehr geringen Konzentrationen bei Al488. D2O kann eingesetzt werden, wenn eine Probe schnell vermessen werden muss, wie zum Beispiel bei Lebendzellmessungen. Nicht nur eine zu niedrige Photonenzahl, auch eine zu geringe Photoschaltrate kann Artefakte bei dSTORM-Messungen erzeugen. Dies wurde anhand von verschiedenen biologischen Strukturen, die mit unterschiedlichen Anregungsintensit{\"a}ten aufgenommen wurden, deutlich gemacht. Besonders die Aufnahmen von Plasmamembranen sind anf{\"a}llig f{\"u}r die Generierung von Artefakten. Sie weisen viele inhomogene und lokal dichte Regionen auf. Wenn nun mehr als ein Emitter pro µm² gleichzeitig an ist, erzeugt das Auswertungsprogramm große artifizielle Cluster. Die hier durchgef{\"u}hrten Messungen machen deutlich, wie wichtig es ist, dSTORM-Bilder immer auf m{\"o}gliche Artefakte hin zu untersuchen, besonders wenn Molek{\"u}le quantifiziert werden sollen. Daf{\"u}r m{\"u}ssen die unbearbeiteten Rohdaten sorgf{\"a}ltig gesichtet werden und notfalls die Messungen mit einer h{\"o}heren Laserleistung wiederholt werden. Da dSTORM mittlerweile immer mehr zur Quantifizierung eingesetzt wird und Clusteranalysen durchgef{\"u}hrt werden, w{\"a}re es sinnvoll bei Ver{\"o}ffentlichungen die Rohdaten von entscheidenden Aufnahmen der {\"O}ffentlichkeit zur Verf{\"u}gung zu stellen. Die F{\"a}rbemethode ist ein weiterer Punkt, durch den Artefakte bei der Abbildung von Molek{\"u}len mittels SMLM entstehen k{\"o}nnen. H{\"a}ufig werden Antik{\"o}rper zum Markieren verwendet. Dabei sollte darauf geachtet werden, dass m{\"o}glichst kleine Antik{\"o}rper oder Antik{\"o}rperfragmente verwendet werden, besonders wenn Clusteranalysen durchgef{\"u}hrt werden sollen. Anderenfalls leidet die Aufl{\"o}sung darunter, bzw. erh{\"o}ht sich die Gefahr der Kreuzvernetzung von Molek{\"u}len. Im zweiten Teil der vorliegenden Arbeit, wurden Plasmamembran-Ceramide untersucht. Ceramide geh{\"o}ren zu den Sphingolipiden und regulieren diverse zellul{\"a}re Prozesse. Verschiedene Stimuli bewirken eine Aktivierung von Sphingomyelinasen (SMasen), die Ceramide in der Plasmamembran synthetisieren. Steigt die Konzentration von Ceramiden in der Plasmamembran an, kondensieren diese zu Ceramid-reichen Plattformen (CRPs). Bisher ist noch wenig {\"u}ber die Verteilung der Ceramide und die Gr{\"o}ße der CRPs bekannt. Sie wurden hier {\"u}ber IgG-Antik{\"o}rper in der Plasmamembran von Jurkat-, U2OS-, HBME- und prim{\"a}ren T-Zellen angef{\"a}rbt und erstmals mit dSTORM hochaufgel{\"o}st, um sie dann zu quantifizieren. Unabh{\"a}ngig von der Zelllinie befanden sich 50\% aller Ceramidmolek{\"u}le in ~ 75 nm großen CRPs. Im Mittel bestanden die CRPs aus ~ 20 Ceramiden. Mit Hilfe einer Titrationsreihe konnte ausgeschlossen werden, dass diese Cluster nur durch die Antik{\"o}rper-F{\"a}rbung artifiziell erzeugt wurden. Bei Inkubation der Zellen mit Bacillus cereus Sphingomyelinase (bSMase) stieg die Gesamtkonzentration der Ceramide in der Plasmamembran an, ebenso wie die Ceramidanzahl innerhalb der CRPs, außerdem die Anzahl und Gr{\"o}ße der CRPs. Dies k{\"o}nnte zu einer Ver{\"a}nderung der L{\"o}slichkeit von Membrankomponenten f{\"u}hren, was wiederum eine Akkumulation bestimmter Rezeptoren oder eine Kompartimentierung bestimmter Proteine erleichtern k{\"o}nnte. Die Anh{\"a}ufung der Ceramide in den CRPs k{\"o}nnte ebenfalls die lokale Interaktion mit anderen Membranmolek{\"u}len erleichtern und dadurch m{\"o}glicherweise die Reaktivit{\"a}t von Rezeptoren ver{\"a}ndern. Mittels Azid-modifizierten Ceramidanaloga und kupferfreier Click-Chemie wurden Plasmamembran-Ceramide auch in lebenden Jurkat-Zellen mit Hilfe konfokaler Laser-Raster-Mikroskopie (CLSM, engl. confocal laser scanning microscopy) und Strukturierter Beleuchtungsmikroskopie (SIM, engl. structured illumination microscopy) untersucht. Dabei konnte gezeigt werden, dass die Fetts{\"a}ure-Kettenl{\"a}nge und die Position des Azids bei den Ceramidanaloga eine entscheidende Rolle spielt, wie hoch das detektierte Signal in der Plasmamembran letztendlich ist. Die Versuche machen auch deutlich, dass die klickbaren Ceramidanaloga lebendzellkompatibel sind, sodass sie eine hervorragende M{\"o}glichkeit darstellen, zellul{\"a}re Reaktionen zu verfolgen. Es wurden hier nicht nur Ceramide in eukaryotischen Zellen analysiert, sondern auch in Bakterien. Neisseria meningitidis (N. meningitidis) sind gramnegative Bakterien, die im Menschen eine Sepsis oder eine Meningitis ausl{\"o}sen k{\"o}nnen. Es wurde mittels immunhistochemischen F{\"a}rbungen mit dem anti-Ceramid IgG-Antik{\"o}rper, aber auch mit den klickbaren Ceramidanaloga, ein Signal in der Membran erhalten, was mit dSTORM hochaufgel{\"o}st wurde. In anderen Bakterien wurden ebenfalls schon Sphingolipide nachgewiesen. Studien zu Ceramiden in N. meningitidis wurden bisher jedoch noch nicht ver{\"o}ffentlicht. Im Rahmen dieser Arbeit konnten erstmals Ergebnisse erhalten werden, die darauf hinweisen, dass N. meningitidis ebenfalls Ceramide besitzen k{\"o}nnten. In einem dritten Projekt wurde die Interaktion zwischen NK-Zellen und Aspergillus fumigatus untersucht. Der Schimmelpilz kann eine Invasive Aspergillose in immunsupprimierten Menschen ausl{\"o}sen, was zum Tod f{\"u}hren kann. Verschiedene Studien konnten schon zeigen, dass NK-Zellen eine wichtige Rolle bei der Bek{\"a}mpfung des Pilzes spielen. Der genaue Mechanismus ist jedoch noch unbekannt. Im Rahmen dieser Arbeit konnte nachgewiesen werden, dass der NK-Zell-Marker CD56 entscheidend f{\"u}r die Pilzerkennung ist. Mit immunhistochemischen F{\"a}rbungen und LSM-, aber auch dSTORM-Messungen, konnte gezeigt werden, dass die normalerweise homogen verteilten CD56-Rezeptoren auf der Plasmamembran von NK-Zellen aktiv an die Interaktionsstelle zu A. fumigatus transportiert werden. Mit der Zeit akkumulieren hier immer mehr CD56-Proteine, w{\"a}hrend das Signal in der restlichen Membran immer weiter abnimmt. Es konnte erstmals CD56 als wichtiger Erkennungsrezeptor f{\"u}r A. fumigatus identifiziert werden. In dem letzten bearbeiteten Projekt, wurde die Bindung von Anti-N-Methyl-D-Aspartat (NMDA)-Rezeptor Enzephalitis Autoantik{\"o}rper an Neuronen untersucht. Bei einer Anti-NMDA-Rezeptor Enzephalitis bilden die Patienten Autoantik{\"o}rper gegen die NR1-Untereinheit ihrer eigenen postsynaptischen NMDA-Rezeptoren. Da die Krankheit oft sehr sp{\"a}t erkannt wird und die Behandlungsm{\"o}glichkeiten noch sehr eingeschr{\"a}nkt sind, f{\"u}hrt sie noch oft zum Tod. Sie wurde erst vor wenigen Jahren beschrieben, sodass der genaue Mechanismus noch unbekannt ist. Im Rahmen dieser Arbeit, konnten erste F{\"a}rbungen mit aufgereinigten Antik{\"o}rper aus Anti-NMDA-Rezeptor Enzephalitis Patienten an NMDA-Rezeptor-transfizierte HEK-Zellen und hippocampalen Maus-Neuronen durchgef{\"u}hrt und mit dSTORM hochaufgel{\"o}st werden. Mit den Messungen der HEK-Zellen konnte best{\"a}tigt werden, dass die Autoantik{\"o}rper an die NR1-Untereinheit der Rezeptoren binden. Es konnten erstmals auch die Bindung der Antik{\"o}rper an Neuronen hochaufgel{\"o}st werden. Dabei wurde sichtbar, dass die Antik{\"o}rper zum einen dicht gepackt in den Synapsen vorliegen, aber auch d{\"u}nner verteilt in den extrasynaptischen Regionen. Basierend auf der Ripley's H-Funktion konnten in den Synapsen große Cluster von ~ 90 nm Durchmesser und im Mittel ~ 500 Lokalisationen und extrasynaptisch kleinere Cluster mit einem durchschnittlichen Durchmesser von ~ 70 nm und ~ 100 Lokalisationen ausgemacht werden. Diese ersten Ergebnisse legen den Grundstein f{\"u}r weitere Messungen, mit denen der Mechanismus der Krankheit untersucht werden kann.},
  subject      = {Ceramide},
  language  = {de}
}
@phdthesis{Aufmkolk2018,
  author    = {Aufmkolk, Sarah},
  title     = {Super-Resolution Microscopy of Synaptic Proteins},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-151976},
  school      = {Universit{\"a}t W{\"u}rzburg},
  pages     = {X, 97},
  year      = {2018},
  abstract  = {The interaction of synaptic proteins orchestrate the function of one of the most complex organs, the brain. The multitude of molecular elements influencing neurological correlations makes imaging processes complicated since conventional fluorescence microscopy methods are unable to resolve structures beyond the diffraction-limit. The implementation of super-resolution fluorescence microscopy into the field of neuroscience allows the visualisation of the fine details of neural connectivity. The key element of my thesis is the super-resolution technique dSTORM (direct Stochastic Optical Reconstruction Microscopy) and its optimisation as a multi-colour approach. Capturing more than one target, I aim to unravel the distribution of synaptic proteins with nanometer precision and set them into a structural and quantitative context with one another. Therefore dSTORM specific protocols are optimized to serve the peculiarities of particular neural samples. In one project the brain derived neurotrophic factor (BDNF) is investigated in primary, hippocampal neurons. With a precision beyond 15 nm, preand post-synaptic sites can be identified by staining the active zone proteins bassoon and homer. As a result, hallmarks of mature synapses can be exhibited. The single molecule sensitivity of dSTORM enables the measurement of endogenous BDNF and locates BDNF granules aligned with glutamatergic pre-synapses. This data proofs that hippocampal neurons are capable of enriching BDNF within the mature glutamatergic pre-synapse, possibly influencing synaptic plasticity. The distribution of the metabotropic glutamate receptor mGlu4 is investigated in physiological brain slices enabling the analysis of the receptor in its natural environment. With dual-colour dSTORM, the spatial arrangement of the mGlu4 receptor in the pre-synaptic sites of parallel fibres in the molecular layer of the mouse cerebellum is visualized, as well as a four to six-fold increase in the density of the receptor in the active zone compared to the nearby environment. Prior functional measurements show that metabotropic glutamate receptors influence voltage-gated calcium channels and proteins that are involved in synaptic vesicle priming. Corresponding dSTORM data indeed suggests that a subset of the mGlu4 receptor is correlated with the voltage-gated calcium channel Cav2.1 on distances around 60 nm. These results are based on the improvement of the direct analysis of localisation data. Tools like coordinated based correlation analysis and nearest neighbour analysis of clusters centroids are used complementary to map protein connections of the synapse. Limits and possible improvements of these tools are discussed to foster the quantitative analysis of single molecule localisation microscopy data. Performing super-resolution microscopy on complex samples like brain slices benefits from a maximised field of view in combination with the visualisation of more than two targets to set the protein of interest in a cellular context. This challenge served as a motivation to establish a workflow for correlated structured illumination microscopy (SIM) and dSTORM. The development of the visualisation software coSIdSTORM promotes the combination of these powerful super-resolution techniques even on separated setups. As an example, synapses in the cerebellum that are affiliated to the parallel fibres and the dendrites of the Purkinje cells are identified by SIM and the protein bassoon of those pre-synapses is visualised threedimensionally with nanoscopic precision by dSTORM. In this work I placed emphasis on the improvement of multi-colour super-resolution imaging and its analysing tools to enable the investigation of synaptic proteins. The unravelling of the structural arrangement of investigated proteins supports the building of a synapse model and therefore helps to understand the relation between structure and function in neural transmission processes.},
  subject      = {Hochaufl{\"o}sende Mikroskopie},
  language  = {en}
}