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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.
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.
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 Würzburg, 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.
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.
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.
Sharpening super-resolution by single molecule localization microscopy in front of a tuned mirror
(2020)
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.
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.
Accurate information transfer between neurons governs proper brain function. At chemical synapses, communication is mediated via neurotransmitter release from specialized presynaptic intercellular contact sites, so called active zones. Their molecular composition constitutes a precisely arranged framework that sets the stage for synaptic communication.
Active zones contain a variety of proteins that deliver the speed, accuracy and plasticity inherent to neurotransmission. Though, how the molecular arrangement of these proteins influences active zone output is still ambiguous. Elucidating the nanoscopic organization of AZs has been hindered by the diffraction-limited resolution of conventional light microscopy, which is insufficient to resolve the active zone architecture on the nanometer scale. Recently, super-resolution techniques entered the field of neuroscience, which yield the capacity to bridge the gap in resolution between light and electron microscopy without losing molecular specificity. Here, localization microscopy methods are of special interest, as they can potentially deliver quantitative information about molecular distributions, even giving absolute numbers of proteins present within cellular nanodomains.
This thesis puts forward an approach based on conventional immunohistochemistry to quantify endogenous protein organizations in situ by employing direct stochastic optical reconstruction microscopy (dSTORM). Focussing on Bruchpilot (Brp) as a major component of Drosophila active zones, the results show that the cytomatrix at the active zone is composed of units, which comprise on average ~137 Brp molecules, most of which are arranged in approximately 15 heptameric clusters. To test for a quantitative relationship between active zone ultrastructure and synaptic output, Drosophila mutants and electrophysiology were employed. The findings indicate that the precise spatial arrangement of Brp reflects properties of short-term plasticity and distinguishes distinct mechanistic causes of synaptic depression. Moreover, functional diversification could be connected to a heretofore unrecognized ultrastructural gradient along a Drosophila motor neuron.
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.
In acute graft-versus-host disease (GVHD) alloreactive donor T cells selectively damage skin, liver, and the gastrointestinal tract while other organs are rarely affected. The mechanism of this selective target tissue infiltration is not well understood. We investigated the importance of alloantigen expression for the selective organ manifestation by examining spatiotemporal changes of cellular and molecular events after allogeneic hematopoietic cell transplantation (allo-HCT). To accomplish this we established a novel multicolor light sheet fluorescence microscopy (LSFM) approach for deciphering immune processes in large tissue specimens on a single-cell level in 3 dimensions. We combined and optimized protocols for antibody penetration, tissue clearing, and triple-color illumination to create a method for analyzing intact mouse and human tissues. This approach allowed us to successfully quantify changes in expression patterns of mucosal vascular addressin cell adhesion molecule–1 (MAdCAM-1) and T cell responses in Peyer’s patches following allo-HCT. In addition, we proofed that LSFM is suitable to map individual T cell subsets after HCT and detected rare cellular events. We employed this versatile technique to study the role of alloantigen expression for the selective organ manifestation after allo-HCT. Therefore, we used a T cell receptor (TCR) transgenic mouse model of GVHD that targets a single peptide antigen and thereby mimics a major histocompatibility complex (MHC)-matched single antigen mismatched (miHAg-mismatched) HCT. We transplanted TCR transgenic (OT-I) T cells into myeloablatively conditioned hosts that either express the peptide antigen ovalbumin ubiquitously (βa-Ova) or selectively in the pancreas (RIP-mOva), an organ that is normally not affected by acute GVHD. Of note, at day+6 after HCT we observed that OT-I T cell infiltration occurred in an alloantigen dependent manner. In βa-Ova recipients, where antigen was ubiquitously expressed, OT-I T cells infiltrated all organs and were not restricted to gastrointestinal tract, liver, and skin. In RIP-mOva recipients, where cognate antigen was only expressed in the pancreas, OT-I T cells selectively infiltrated this organ that is usually spared in acute GVHD. In conditioned RIP-mOva the transfer of 100 OT-I T cells sufficed to effectively infiltrate and destroy pancreatic islets resulting in 100% mortality. By employing intact tissue LSFM in RIP-mOva recipients, we identified very low numbers of initial islet infiltrating T cells on day+4 after HCT followed by a massive T cell migration to the pancreas within the following 24 hours. This suggested an effective mechanism of effector T cell recruitment to the tissue of alloantigen expression after initial antigen specific T cell encounter. In chimeras that either expressed the model antigen ovalbumin selectively in hematopoietic or in parenchymal cells only, transplanted OT-I T cells infiltrated target tissues irrespective of which compartment expressed the alloantigen. As IFN-γ could be detected in the serum of transplanted ovalbumin expressing recipients (βa-Ova, βa-Ova-chimeras and RIP-mOva) at day+6 after HCT, we hypothesized that this cytokine may be functionally involved in antigen specific OT-I T cell mediated pathology. In vitro activated OT-I T cells responded with the production of IFN-γ upon antigen re-encounter suggesting that IFN-γ might be relevant in the alloantigen dependent organ infiltration of antigen specific CD8+ T cell infiltration after HCT. Based on these data we propose that alloantigen expression plays an important role in organ specific T cell infiltration during acute GVHD and that initial alloreactive T cells recognizing the cognate antigen propagate a vicious cycle of enhanced T cell recruitment that subsequently culminates in the exacerbation of tissue restricted GVHD.