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Theoretical and experimental concepts to increase the performance of structured illumination microscopyStröhl, Florian January 2018 (has links)
The aim of the work described in this thesis is to improve the understanding, implementation, and overall capabilities of structured illumination microscopy (SIM). SIM is a superresolution technique that excels in gentle live-cell volumetric imaging tasks. Many modalities of SIM were developed over the last decade that tailored SIM into the versatile and powerful technique that it is today. Nevertheless, the field of SIM continues to evolve and there is plenty of room for novel concepts. Specifically, in this thesis, a generalised framework for a theoretical description of SIM variants is introduced, the constraints of optical components for a flexible SIM system are discussed and the set-up is realised, the important aspect of deconvolution in SIM is highlighted and further developed, and finally novel SIM modalities introduced that improve its time-resolution, gentleness, and volumetric imaging capabilities. Based on the generalised theory, the computational steps for the extraction of superresolution information from SIM raw data are outlined and the essential concept of spatial frequency un-mixing explained for standard SIM as well as for multifocal SIM. Multifocal SIM hereby acts as a parallelised confocal as well as widefield technique and thus serves as link between the two modalities. Using this novel scheme deconvolution methods for SIM are then further developed to allow a holistic reconstruction procedure. Deconvolution is of great important in the SIM reconstruction process, and hence rigorous derivations of advanced deconvolution methods are provided and further developed to enable generalised ‘multi-image’ Richardson-Lucy deconvolution in SIM, called joint Richardson-Lucy deconvolution (jRL). This approach is demonstrated to robustly produce optically sectioned multifocal SIM images and, through the incorporation of a 3D imaging model, also volumetric standard SIM images within the jRL framework. For standard SIM this approach enabled acquisition speed doubling, because the recovery of superresolved images from a reduced number of raw frames through constrained jRL was made possible. The method is validated in silico and in vitro. For the study of yet faster moving samples deconvolution microscopy is found to be the method of choice. To enable optical sectioning, a key feature of SIM, in deconvolution microscopy, a new modality of optical sectioning microscopy is introduced that can be implemented as a single-shot technique. Via polarised excitation and detection in orthogonal directions in conjunction with structured illumination the theoretical framework is rigorously derived and validated.
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Micro-Anatomical Quantitative Imaging Towards Enabling Automated Diagnosis of Thick Tissues at the Point of CareMueller, Jenna Lynne Hook January 2015 (has links)
<p>Histopathology is the clinical standard for tissue diagnosis. However, histopathology has several limitations including that it requires tissue processing, which can take 30 minutes or more, and requires a highly trained pathologist to diagnose the tissue. Additionally, the diagnosis is qualitative, and the lack of quantitation leads to possible observer-specific diagnosis. Taken together, it is difficult to diagnose tissue at the point of care using histopathology.</p><p>Several clinical situations could benefit from more rapid and automated histological processing, which could reduce the time and the number of steps required between obtaining a fresh tissue specimen and rendering a diagnosis. For example, there is need for rapid detection of residual cancer on the surface of tumor resection specimens during excisional surgeries, which is known as intraoperative tumor margin assessment. Additionally, rapid assessment of biopsy specimens at the point-of-care could enable clinicians to confirm that a suspicious lesion is successfully sampled, thus preventing an unnecessary repeat biopsy procedure. Rapid and low cost histological processing could also be potentially useful in settings lacking the human resources and equipment necessary to perform standard histologic assessment. Lastly, automated interpretation of tissue samples could potentially reduce inter-observer error, particularly in the diagnosis of borderline lesions. </p><p>To address these needs, high quality microscopic images of the tissue must be obtained in rapid timeframes, in order for a pathologic assessment to be useful for guiding the intervention. Optical microscopy is a powerful technique to obtain high-resolution images of tissue morphology in real-time at the point of care, without the need for tissue processing. In particular, a number of groups have combined fluorescence microscopy with vital fluorescent stains to visualize micro-anatomical features of thick (i.e. unsectioned or unprocessed) tissue. However, robust methods for segmentation and quantitative analysis of heterogeneous images are essential to enable automated diagnosis. Thus, the goal of this work was to obtain high resolution imaging of tissue morphology through employing fluorescence microscopy and vital fluorescent stains and to develop a quantitative strategy to segment and quantify tissue features in heterogeneous images, such as nuclei and the surrounding stroma, which will enable automated diagnosis of thick tissues.</p><p>To achieve these goals, three specific aims were proposed. The first aim was to develop an image processing method that can differentiate nuclei from background tissue heterogeneity and enable automated diagnosis of thick tissue at the point of care. A computational technique called sparse component analysis (SCA) was adapted to isolate features of interest, such as nuclei, from the background. SCA has been used previously in the image processing community for image compression, enhancement, and restoration, but has never been applied to separate distinct tissue types in a heterogeneous image. In combination with a high resolution fluorescence microendoscope (HRME) and a contrast agent acriflavine, the utility of this technique was demonstrated through imaging preclinical sarcoma tumor margins. Acriflavine localizes to the nuclei of cells where it reversibly associates with RNA and DNA. Additionally, acriflavine shows some affinity for collagen and muscle. SCA was adapted to isolate acriflavine positive features or APFs (which correspond to RNA and DNA) from background tissue heterogeneity. The circle transform (CT) was applied to the SCA output to quantify the size and density of overlapping APFs. The sensitivity of the SCA+CT approach to variations in APF size, density and background heterogeneity was demonstrated through simulations. Specifically, SCA+CT achieved the lowest errors for higher contrast ratios and larger APF sizes. When applied to tissue images of excised sarcoma margins, SCA+CT correctly isolated APFs and showed consistently increased density in tumor and tumor + muscle images compared to images containing muscle. Next, variables were quantified from images of resected primary sarcomas and used to optimize a multivariate model. The sensitivity and specificity for differentiating positive from negative ex vivo resected tumor margins was 82% and 75%. The utility of this approach was further tested by imaging the in vivo tumor cavities from 34 mice after resection of a sarcoma with local recurrence as a bench mark. When applied prospectively to images from the tumor cavity, the sensitivity and specificity for differentiating local recurrence was 78% and 82%. The results indicate that SCA+CT can accurately delineate APFs in heterogeneous tissue, which is essential to enable automated and rapid surveillance of tissue pathology. </p><p>Two primary challenges were identified in the work in aim 1. First, while SCA can be used to isolate features, such as APFs, from heterogeneous images, its performance is limited by the contrast between APFs and the background. Second, while it is feasible to create mosaics by scanning a sarcoma tumor bed in a mouse, which is on the order of 3-7 mm in any one dimension, it is not feasible to evaluate an entire human surgical margin. Thus, improvements to the microscopic imaging system were made to (1) improve image contrast through rejecting out-of-focus background fluorescence and to (2) increase the field of view (FOV) while maintaining the sub-cellular resolution needed for delineation of nuclei. To address these challenges, a technique called structured illumination microscopy (SIM) was employed in which the entire FOV is illuminated with a defined spatial pattern rather than scanning a focal spot, such as in confocal microscopy. </p><p>Thus, the second aim was to improve image contrast and increase the FOV through employing wide-field, non-contact structured illumination microscopy and optimize the segmentation algorithm for new imaging modality. Both image contrast and FOV were increased through the development of a wide-field fluorescence SIM system. Clear improvement in image contrast was seen in structured illumination images compared to uniform illumination images. Additionally, the FOV is over 13X larger than the fluorescence microendoscope used in aim 1. Initial segmentation results of SIM images revealed that SCA is unable to segment large numbers of APFs in the tumor images. Because the FOV of the SIM system is over 13X larger than the FOV of the fluorescence microendoscope, dense collections of APFs commonly seen in tumor images could no longer be sparsely represented, and the fundamental sparsity assumption associated with SCA was no longer met. Thus, an algorithm called maximally stable extremal regions (MSER) was investigated as an alternative approach for APF segmentation in SIM images. MSER was able to accurately segment large numbers of APFs in SIM images of tumor tissue. In addition to optimizing MSER for SIM image segmentation, an optimal frequency of the illumination pattern used in SIM was carefully selected because the image signal to noise ratio (SNR) is dependent on the grid frequency. A grid frequency of 31.7 mm-1 led to the highest SNR and lowest percent error associated with MSER segmentation. </p><p>Once MSER was optimized for SIM image segmentation and the optimal grid frequency was selected, a quantitative model was developed to diagnose mouse sarcoma tumor margins that were imaged ex vivo with SIM. Tumor margins were stained with acridine orange (AO) in aim 2 because AO was found to stain the sarcoma tissue more brightly than acriflavine. Both acriflavine and AO are intravital dyes, which have been shown to stain nuclei, skeletal muscle, and collagenous stroma. A tissue-type classification model was developed to differentiate localized regions (75x75 µm) of tumor from skeletal muscle and adipose tissue based on the MSER segmentation output. Specifically, a logistic regression model was used to classify each localized region. The logistic regression model yielded an output in terms of probability (0-100%) that tumor was located within each 75x75 µm region. The model performance was tested using a receiver operator characteristic (ROC) curve analysis that revealed 77% sensitivity and 81% specificity. For margin classification, the whole margin image was divided into localized regions and this tissue-type classification model was applied. In a subset of 6 margins (3 negative, 3 positive), it was shown that with a tumor probability threshold of 50%, 8% of all regions from negative margins exceeded this threshold, while over 17% of all regions exceeded the threshold in the positive margins. Thus, 8% of regions in negative margins were considered false positives. These false positive regions are likely due to the high density of APFs present in normal tissues, which clearly demonstrates a challenge in implementing this automatic algorithm based on AO staining alone. </p><p>Thus, the third aim was to improve the specificity of the diagnostic model through leveraging other sources of contrast. Modifications were made to the SIM system to enable fluorescence imaging at a variety of wavelengths. Specifically, the SIM system was modified to enabling imaging of red fluorescent protein (RFP) expressing sarcomas, which were used to delineate the location of tumor cells within each image. Initial analysis of AO stained panels confirmed that there was room for improvement in tumor detection, particularly in regards to false positive regions that were negative for RFP. One approach for improving the specificity of the diagnostic model was to investigate using a fluorophore that was more specific to staining tumor. Specifically, tetracycline was selected because it appeared to specifically stain freshly excised tumor tissue in a matter of minutes, and was non-toxic and stable in solution. Results indicated that tetracycline staining has promise for increasing the specificity of tumor detection in SIM images of a preclinical sarcoma model and further investigation is warranted. </p><p>In conclusion, this work presents the development of a combination of tools that is capable of automated segmentation and quantification of micro-anatomical images of thick tissue. When compared to the fluorescence microendoscope, wide-field multispectral fluorescence SIM imaging provided improved image contrast, a larger FOV with comparable resolution, and the ability to image a variety of fluorophores. MSER was an appropriate and rapid approach to segment dense collections of APFs from wide-field SIM images. Variables that reflect the morphology of the tissue, such as the density, size, and shape of nuclei and nucleoli, can be used to automatically diagnose SIM images. The clinical utility of SIM imaging and MSER segmentation to detect microscopic residual disease has been demonstrated by imaging excised preclinical sarcoma margins. Ultimately, this work demonstrates that fluorescence imaging of tissue micro-anatomy combined with a specialized algorithm for delineation and quantification of features is a means for rapid, non-destructive and automated detection of microscopic disease, which could improve cancer management in a variety of clinical scenarios.</p> / Dissertation
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Optical techniques for the investigation of a mechanical role for FRMD6/Willin in the Hippo signalling pathwayGoff, Frances January 2019 (has links)
The mammalian hippo signalling pathway controls cell proliferation and apoptosis via transcriptional co-activators YAP and TAZ, and as such is a key regulator of organ and tissue growth. Multiple cellular components converge in this pathway, including the actin cytoskeleton, which is required for YAP/TAZ activity. The precise mechanism by which the mechanical actomyosin network regulates Hippo signalling, however, is unknown. Optical methods provide a non-invasive way to image and study the biomechanics of cells. In the past two decades, super-resolution fluorescence microscopy techniques that break the diffraction limit of light have come to the fore, enabling visualisation of intracellular detail at the nanoscale level. Optical trapping, on the other hand, allows precise control of micron-sized objects such as cells. Here, super resolution structured illumination microscopy (SIM) and elastic resonator interference stress microscopy (ERISM) were used to investigate a potential role for the FERM-domain protein FRMD6, or Willin, in the mechanical control of the Hippo pathway in a neuronal cell model. A double optical trap was also integrated with the Nikon-SIM with the aim of cell stretching. Willin expression was shown to modify the morphology, neuronal differentiation, actin cytoskeleton and forces of SH-SY5Y cells. Optical trapping from above the SIM objective, however, was demonstrated to be ineffective for manipulation of adherent cells. The results presented here indicate a function for Willin in the assembly of actin stress fibres that may be the result of an interaction with the Hippo pathway regulator AMOT. Further investigation, for example by direct cell stretching, is required to elucidate the exact role of Willin in the mechanical control of YAP/TAZ.
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The genetic regulation and subcellular dynamics of secretory and endolysosomal organelles of Drosophila secondary cellsKroeger, Benjamin Robert January 2017 (has links)
Secretory processes underpin the emergence of cellular diversity in complex multicellular organisms. However, our understanding of the basic mechanisms controlling the different secretory and endosomal compartments involved remains surprisingly incomplete. During my DPhil I have studied a specialised epithelial cell type in the male Drosophila accessory glands, the secondary cell, which contains unusually large intracellular compartments that are accessible to detailed morphological study. I characterise the organisation, ultrastructure and molecular composition of this cell's secretory and endosomal compartments, and I employ specific Rab GTPases, conserved coordinators of membrane trafficking and identity, to define multiple compartmental subtypes. By developing super-resolution and time-lapse microscopy approaches in these cells, I show that numerous intraluminal vesicles (ILVs) are formed within Rab11-labelled secretory compartments and released into the accessory gland lumen as exosomes, the first clear demonstration in eukaryotic cells of exosome biogenesis within a non-late endosomal compartment. Biogenesis of these ILVs is dependent on evolutionarily conserved Endosomal Sorting Complexes Required for Transport (ESCRT) 0-III genes and involves loading of compartment-specific cargoes. Work by others, some in collaboration with me, has shown that these novel mechanisms are conserved in human cells. I show that dense-core granules, the structures employed to package proteins and other molecules destined for regulated secretion, form within large non-cored Rab6- positive compartments, in a process that seems to involve inputs from both the Golgi and recycling endosomal pathways. Further analysis has revealed roles for specific Rabs, for ILVs, and for the conserved fibrillar protein Mfas/TGFBI in different aspects of DCG formation. I also show that DCGs are not only secreted, but can also be degraded by fusion to acidic endosomal compartments. Remarkably, there is evidence that mammalian cells may employ all of these mechanisms and defects in these processes may be linked to diseases like cancer, diabetes and neurodegenerative disorders. Hence my work has established a new system to study complex secretory mechanisms, which can now be developed to model specific disease processes in the future. In summary, I have discovered several novel cell biological mechanisms controlling exosome biology, dense-core granule biogenesis, regulated secretion, and endolysosomal trafficking. Some of these already appear relevant to human health and disease, suggesting that the secondary cell system has considerable further potential for unravelling the fundamental processes underlying eukaryotic secretion in the future.
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Advanced light-sheet and structured illumination microscopy techniques for neuroscience and disease diagnosisNylk, Jonathan January 2016 (has links)
Optical microscopy is a cornerstone of biomedical research. Advances in optical techniques enable specific, high resolution, sterile, and biologically compatible imaging. In particular, beam shaping has been used to tailor microscopy techniques to enhance microscope performance. The aim of this Thesis is to investigate the use of novel beam shaping techniques in emerging optical microscopy methods, and to apply these methods in biomedicine. To overcome the challenges associated with high resolution imaging of large specimens, the use of Airy beams and related techniques are applied to light-sheet microscopy. This approach increases the field-of-view that can be imaged at high resolution by over an order of magnitude compared to standard Gaussian beam based light-sheet microscopy, has reduced phototoxicity, and can be implemented with a low-cost optical system. Advanced implementations show promise for imaging at depth within turbid tissue, in particular for neuroscience. Super-resolution microscopy techniques enhance the spatial resolution of optical methods. Structured illumination microscopy is investigated as an alternative for electron microscopy in disease diagnosis, capable of visualising pathologically relevant features of kidney disease. Separately, compact optical manipulation methods are developed with the aim of adding functionality to super-resolution techniques.
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Microscopie de fluorescence rapide et optique adaptative pour l'étude fonctionnelle tridimensionnelle in vivo des réseaux neuronaux impliqués dans la mémoire chez Drosophila melanogaster / Fast fluorescence microscopy and adaptive optics for in vivo tridimensional functional imaging of neural circuits involved in memory formation in Drosophila MelanogasterPedrazzani, Mélanie 14 December 2015 (has links)
L’utilisation de techniques de microscopie optique de plus en plus performantes a permis des avancées considérables en neurobiologie. Néanmoins, la population de neurones mise en jeu lors de la formation de la mémoire, ainsi que sa dynamique restent à ce jour très peu connues. L’objectif de la thèse est de développer puis d’utiliser deux types de microscopies originales couplées à des sondes fluorescentes de dernière génération (sondes calciques G-CaMP6f et sonde voltage ArcLight) pour l’étude in vivo des réseaux neuronaux impliqués dans la mémorisation chez la drosophile. Le choix du modèle Drosophila melanogaster pour cette étude neurobiologique est justifié par plusieurs atouts uniques : un cerveau peu volumineux, des capacités d’apprentissage remarquables, la possibilité d’analyser un réseau neuronal dans sa globalité avec une résolution cellulaire et la disponibilité d’outils génétiques très perfectionnés pour son étude. Le premier type de microscopie conçu est celui dite à illumination structurée de type HiLo permettant d'obtenir une coupe optique en profondeur. Nous avons alors étudié le rôle de divers récepteurs, tels que les récepteurs dopaminergiques et gabaergiques dans la transmission de l'information punitive jusqu'aux neurones des corps pédonculés. Nous avons également mis en évidence une non-homogénéité spatiale des neurones de type α/β des corps pédonculés en termes d’excitation et d’inhibition en réponse à une stimulation punitive pour une profondeur d’analyse d'environ 10 à 20 µm. Cette profondeur limite étant imposée par les aberrations, nous avons alors implémenté une boucle d’optique adaptative dans notre microscope. Cela a permis de réaliser des analyses morphologiques jusqu’à 50 µm de profondeur. Le second type de microscopie développé est la microscopie multiconfocale de type « spinning disk » dans le but d’imager l’ensemble des corps cellulaires des neurones des corps pédonculés. Le développement de ce projet n'est pas achevé, ce qui n’a pas encore permis de répondre à des questions biologiques d’intérêt. / Cellular and neural network dynamics involved in memory formation remain poorly known despite the progress brought by advanced optical microscopies to neurobiology. The use of Drosophila melanogaster as a model organism constitute one of the most promising approaches due to its unique features: a small brain size, outstanding learning capabilities, very powerful genetic tools and the possibility to analyze a whole neural network with a cellular resolution. To this aim, we implemented two types of optical fluorescence microscopes coupled to cutting-edge fluorescent biosensors, calcic G-CaMP6f and voltage ArcLight probes. We used HiLo structured illumination, a technique able to provide axial optical sectioning, deep in the brain, to study the role of dopaminergic and gabaergic molecular receptors in the transmission of aversive stimulus to mushroom bodies neurons. We also evidenced a non-uniform response of type α/β mushroom bodies neurons under electrical stimulation at 10 to 20 µm depth of analysis. To penetrate deeper in the brain, we added an adaptive optics feedback loop into our microscope in order to overcome aberrations issues. We were then able to rebuild optical sections down to 50 µm depth. The second type of microscopy we developed is a multiconfocal microscope using spinning disk. The aim was to image all the mushroom bodies neurons, at the level of their cell bodies, with a cellular resolution. Since this project is at its beginning, it did not allow us to answer to advanced biological questions yet.
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Task-specific uncertainty of areal surface texture measurement using structured illumination microscopyLi, Zhen 31 May 2023 (has links)
Surface quality plays a vital role in controlling the function performance of the workpiece. With the development of the measuring technique, areal surface measurement has been widely applied in the industry. However, estimating the uncertainty of areal surface measurement is still a challenge. Except for the metrological characteristics of the measurement system, measurement conditions should be considered for uncertainty evaluation.
The dissertation investigates the influence of measurement settings on surface measurement. A silver-plated surface, three different rough grinding surfaces, and three different rough cylindrical grinding surfaces were measured using structured illumination microscopy. The measurements were at the different objective lenses, vertical scanning interval, exposure time, and sample tilt. The results show that the measurement settings influence the non-measured points, measurement noise, and areal surface texture parameters. Therefore, according to the investigation, the sample tilt and exposure time should also be included in the uncertainty budget.
An approach was proposed to investigate the influence of non-measured points on the areal surface texture parameters. The relation between the non-measured points ratio and measurement settings was investigated, and how the areal surface texture parameters changed due to the non-measured points was studied. Moreover, an approach based on the metrological characteristic method was proposed to estimate the uncertainty due to the measurement noise. This method can be extended to the uncertainty evaluation due to other metrological characteristics. Additionally, an approach based on the Monte Carlo Method was proposed to estimate the measurement uncertainty due to different influences. This approach was verified as feasible in the practical measurement.
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Optical micro-manipulation in HIV-1 infected cells for improved HIV-1 treatment and diagnosisLugongolo, Masixole Yvonne 06 1900 (has links)
Laser application in the field of biological and medical sciences has significantly grown, thereby
strengthening the field of Biophotonics. Research conducted in Biophotonics focuses on the concept
of using light especially in the visible and near infrared regions of the electromagnetic radiation for
the evaluation of living systems. In this thesis new discoveries are presented about low level laser
therapy, optical trapping, transmission spectroscopy, luminescence spectroscopy and structured
illumination microscopy (SIM), displaying the impact each technique has on HIV infected cells. The
results showed that the irradiation of HIV-1 infected TZM-bl cells with low power red laser reduces
HIV-1 infection. The outcomes of this study further proved that when irradiation is used in
conjunction with efavirenz, an antiretroviral drug, HIV-1 infection could be reduced to undetectable
levels in TZM-bl cells. Through the coupling of transmission spectroscopy with optical trapping, and
separately, use of luminescence spectroscopy, label free diagnosis of HIV in infected cell samples
was achieved. This finding affirms that HIV-1 infection can be detected in a label free manner when
using laser based techniques. Furthermore, the photoluminescence spectrometer system was
employed to generate a decay curve, which was necessary so as to have some understanding on
lifetime of the luminescent signal in infected TZM-bl cells. Finally, in order to confirm that indeed
TZM-bl cells were infected, an established super-resolution microscopy system SIM was used to
detect HIV-1 infection in TZM-bl cells. Indeed in the infected cells viral molecules p24 and gp41
were detected through SIM, while they were not detected in uninfected cells. In future studies, super
resolution microscopy would be coupled to an optical trapping system in order to confirm that each
trapped cells is whether infected or uninfected so as to improve HIV diagnosis. / College of Science, Engineering and Technology / Ph. D. (Science, Engineering and Technology)
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Quantitation Strategies in Optically Sectioning Fluorescence Microscopy / Quantifizierungsstrategien in der optisch schnittbildenden FluoreszenzmikroskopieWeigel, Arwed 15 January 2009 (has links)
No description available.
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Proteins bind Neutrophil extracellular traps in specific patternsWinkler, Jonay Moritz Julius 24 June 2024 (has links)
Neutrophile sind die häufigsten weißen Blutkörperchen im menschlichen Blut. Sie bilden die
erste Verteidigungslinie und töten eindringende Krankheitserreger ab. Neutrophile
extrazelluläre Fallen (NETs) sind netzartige Strukturen, die aus dekondensiertem Chromatin
bestehen und mit zytotoxischen Proteinen dekoriert sind. NETs können Mikroben in vitro
und in vivo einfangen und abtöten, sind aber auch für verschiedene Krankheiten
verantwortlich. Frühere Studien haben eine spezifische Gruppe von 20-50
Neutrophilenproteinen identifiziert, die an NETs gebunden sind und von denen einige eine
mikrobizide Wirkung haben. Wie diese Proteine an die NETs binden, wie sie interagieren
und wie die Bindung ihre antimikrobielle Aktivität beeinflusst, ist noch nicht bekannt.
In dieser Dissertation habe ich die Verteilung von acht neutrophilen Proteinen und
Nukleosomen auf NETs mit Hilfe der Superauflösungsmikroskopie untersucht. Es wurden
drei unabhängige Techniken mit Auflösungen von mehr als 90 nm verwendet. Die
Nukleosomen bildeten auf den NETs periodische Cluster mit deutlich größeren Abständen
im Vergleich zum kondensierten Chromatin. Drei NET-Proteine waren ebenfalls in
periodischen Clustern auf den NETs lokalisiert und zwei von ihnen waren stark mit
Nukleosomen kolokalisiert. Alle anderen analysierten Proteine zeigten keine Muster der
Bindung an NETs. Zusammengenommen zeigen diese Ergebnisse, dass die Bindung von
Proteinen an NETs zumindest teilweise spezifisch ist und teilweise durch Wechselwirkungen
mit Nukleosomen vermittelt wird.
Die erfolgreiche Einführung der superauflösenden Mikroskopie für schwierige NET-Proben
in Kombination mit einem vorgeschlagenen rekonstituierten NET-System eröffnet neue
Möglichkeiten für das Verständnis der molekularen Mechanismen der NET-Bildung und der
Protein-Protein-Interaktion bei der NET-vermittelten Abtötung. / Neutrophils are the most abundant human white blood cell in circulation. They are the first
line of defense and kill invading pathogens. Neutrophil Extracellular Traps (NETs) are weblike
structures composed of decondensed chromatin decorated with cytotoxic proteins. NETs
can trap and kill microbes in vitro and in vivo, but also mediate several diseases. Previous
studies identified a specific set of 20-50 neutrophil proteins bound to NETs, several with
microbicidal activity. It remains unknown how these proteins bind to NETs, how they interact
and how binding influences their anti-microbial activity.
In this dissertation, I studied the distribution of eight neutrophil proteins and nucleosomes
on NETs using super-resolution microscopy. Three independent techniques with resolutions
larger than 90nm were used. Nucleosomes formed periodic clusters on NETs, with
significantly larger spacing compared to condensed chromatin. Three NET proteins also
localized in periodic clusters on NETs and two of them strongly co-localized with
nucleosomes. All other proteins analyzed showed no patterns binding to NETs. Taken
together, these findings demonstrate that, at least some, protein binding to NETs is specific
and in part mediated by interactions with nucleosomes.
The successful introduction of super-resolution microscopy to the challenging NET samples
in combination with a proposed reconstituted NET system opens new possibilities to
understand the molecular mechanisms behind NET formation and protein-protein
interaction in NET mediated killing.
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