356 research outputs found
UC2 – A Versatile and Customizable low-cost 3D-printed Optical Open-Standard for microscopic imaging
No full textThis is a collection of data used in the pre-print "UC2 – A Versatile and Customizable low-cost 3D-printed Optical Open-Standard for microscopic imaging". Additional toolboxes like the "Deconv Toolbox" by Rainer Heintzmann are available upon request
Development of a holoscopic imaging system and applied high-resolution fluorescence microscopy
No full textBiomedical imaging helps extending our comprehension of ourselves and our environment. Advances in camera, laser and computation technologies have enabled an ever-increasing number of imaging technologies. Imaging with visible and infrared light has the advantage that it is less harmful than other radiation and its wavelength is in the order of magnitude of cells and subcellular components. Fluorescence microscopy provides good chemical contrast and multi-colour imaging can help elucidate cellular architecture. Incoherent superresolution methods permit us to bypass Abbe's diffraction limit of lateral resolution and visualize previously unnoticed details. Coherent imaging methods such as optical coherence tomography or holoscopy do not require any previous labelling and have the advantage that they record both the amplitude and phase of the light emitted from a scattering sample by interferometric superposition with a reference wave. Both incoherent an coherent imaging methods are used in this thesis. The results of two interdisciplinary research collaborations using different fluorescence microscopy methods, including superresolution methods, are presented. Podosomes in macrophages were studied with stimulated emission depletion microscopy, structured illumination microscopy and localisation microscopy and a distinctly polygonal shape in their vinculin rings was found. Image processing routines allowed for a quantitative analysis of the acquired images [1]. In the second study, chlorophyll, the most prominent natural pigments, and digested chlorophyll metabolites were detected in gut section of the herbivorous Spodoptera littoralis larva. Widefield and high-resolution autofluorescence microscopy revealed that the brush border membranes of their gut are covered with the chlorophyllide binding protein tightly bound to the gut membrane. A function in defense against gut microbes is discussed [2]. Optical coherence tomography (OCT) offers a slightly lower spatial resolution than light microscopy but generally better penetration depths. In order to use a higher numerical aperture for detection in OCT, the dilemma of the resulting reduced depth of field has to be overcome. Different extended focus possibilities are explored in this thesis. Bessel illumination is an established method to achieve an extended depth of field without compromising the lateral resolution. When broadband or multicolour imaging is required, wavelength-dependent changes in the radial profile of the Bessel illumination can however complicate further image processing and analysis. A solution for engineering a multicolour Bessel beam was implemented with a phase-only spatial light modulator in the image plane and an iterative Fourier Transformation algorithm [3]. For higher acquisition speed, full-field recording is favourable to scanning the scattering sample with a Bessel beam. OCT can be combined with reconstruction methods from digital holography to achieve an extended focus numerically. A suitable experimental imaging setup and a custom-written reconstruction algorithm are presented. [1] M. Walde, J. Monypenny, R. Heintzmann, G. E. Jones, and S. Cox, “Vinculin binding angle in podosomes revealed by high resolution microscopy”, PLoS ONE, vol. 9, no. 2, 2014. [2] A. Badgaa, R. Büchler, N. Wielsch, M. Walde, R. Heintzmann, Y. Pauchet, A. Svatos, K. Ploss, and W. Boland, “The Green Gut: Chlorophyll Degradation in the Gut of Spodoptera littoralis”, Journal of Chemical Ecology, vol. 41, no. 11, pp. 965-974, 2015. [3] M. Walde, A. Jost, K. Wicker, and R. Heintzmann, “Engineering an achromatic Bessel beam using a phase-only spatial light modulator and an iterative Fourier transformation algorithm”, Optics Communications, vol. 383, pp. 64-68, 2017
Super-Resolution Structured Illumination Microscopy
No full textHeintzmann R, Huser T. Super-Resolution Structured Illumination Microscopy. CHEMICAL REVIEWS. 2017;117(23):13890-13908.Super-resolved structured illumination microscopy (SR-SIM) is among the most rapidly growing fluorescence microscopy techniques that can surpass the optical diffraction limit. The strength of SR-SIM is that it can be readily applied to samples prepared for conventional fluorescence microscopy, requiring no sophisticated sample preparation protocols. As an extension of wide-field fluorescence microscopy, it is inherently capable of multicolor imaging and optical sectioning and, with sufficiently fast implementations, permits live cell imaging. Image reconstruction, however, currently relies on sophisticated computational procedures, susceptible to reconstruction artifacts, requiring trained users to recognize and avoid them. Here, we review the latest developments in SR-SIM research. Starting from a historical overview of the development of SR-SIM, we review how this method can be implemented in various experimental schemes, we provide an overview of the important parameters involved in successful image reconstruction, we summarize recent biological applications, and we provide a brief outlook of the directions in which we believe SR-SIM is headed in the future
The SNARE-motif is essential for the formation of syntaxin clusters in the plasma membrane.
No full textAbstractIn the plasma membrane, syntaxin 1 and syntaxin 4 clusters define sites at which secretory granules and caveolae fuse, respectively. It is widely believed that lipid phases are mandatory for cluster formation, as cluster integrity depends on cholesterol. Here we report that the native lipid environment is not sufficient for correct syntaxin 1 clustering and that additional cytoplasmic protein-protein interactions, primarily involving the SNARE motif, are required. Apparently no specific cofactors are needed because i), clusters form equally well in nonneuronal cells, and ii), as revealed by nanoscale subdiffraction resolution provided by STED microscopy, the number of clusters directly depends on the syntaxin 1 concentration. For syntaxin 4 clustering the N-terminal domain and the linker region are also dispensable. Moreover, clustering is specific because in both cluster types syntaxins mutually exclude one another at endogenous levels. We suggest that the SNARE motifs of syntaxin 1 and 4 mediate specific syntaxin clustering by homooligomerization, thereby spatially separating sites for different biological activities. Thus, syntaxin clustering represents a mechanism of membrane patterning that is based on protein-protein interactions
New effects in polynucleotide release from cationic lipid carriers revealed by confocal imaging, fluorescence cross-correlation spectroscopy and single particle tracking
No full textWe report on new insights into the mechanisms of short single and double stranded oligonucleotide release from cationic lipid complexes (lipoplexes), used in gene therapy. Specifically, we modeled endosomal membranes using giant unilamellar vesicles and investigated the roles of various individual cellular phospholipids in interaction with lipoplexes. Our approach uses a combination of confocal imaging, fluorescence cross-correlation spectroscopy and single particle tracking, revealing several new aspects of the release: (a) phosphatidylserine and phosphatidylethanolamine are equally active in disassembling lipoplexes, while phosphatidylcholine and sphingomyelin are inert; (b) in contrast to earlier findings, phosphatidylethanolamine alone, in the absence of anionic phosphatidylserine triggers extensive release; (c) a double-stranded DNA structure remains well preserved after release; (d) lipoplexes exhibited preferential binding to transient lipid domains, which appear at the onset of lipoplex attachment to originally uniform membranes and vanish after initiation of polynucleotide release. The latter effect is likely related to phosphatidyleserine redistribution in membranes due to lipoplex binding. Real time tracking of single DOTAP/DOPE and DOTAP/DOPC lipoplexes showed that both particles remained compact and associated with membranes up to 1–2 min before fusion, indicating that a more complex mechanism, different from suggested earlier rapid fusion, promotes more efficient transfection by DOTAP/DOPE complexes
Modular Open-Source toolbox for optics education
No full textUC2 is an open-source modular toolbox for making hardware projects based on functional optical blocks. It creates a bridge between education and science by offering an alternative to the standard tools, a generic toolbox that can be used for both fields. The same basic hardware is meant to be used for teaching photonics and its applications in an interactive way and the students could later use the same system in their own work for prototyping. As a community-driven collaborative project, UC2 benefits from the experience of its early adopters and from its attractiveness for project-based courses. It is a collective effort of many researchers and students. This thesis shows the contributions of the author to the project. In this work, we present the development and testing of the hardware for the educational applications. The key aspects of open-source hardware design are evaluated and summarized into a decision matrix defining the boundary conditions of the development. Special importance is given to improving accessibility of the toolbox by providing comprehensive documentation and lowering the entry barrier. Four different educational kits, in summary called “TheBOX”, were developed, with the MiniBOX being at the stage of a production-ready prototype, optimized for mass production. The BOXes are comprehensive toolkits aimed at a specific education level or demonstration of certain experiments. These present a low-cost alternative to the commercially available systems. Additionally to the advantage in price, they offer a lower entry level and create an inviting environment for science education. They were developed in close contact with the users and the improvements were based on iteratively acquired feedback. The system was tested in many workshops and courses and provided tools for student projects
Entwicklung einer endoskopischen Fasersonde für optische Biopsien mithilfe nichtlinearer Spektroskopie
No full textEine frühe Erkennung und exakte Diagnostik von Krankheiten wie beispielsweise Tumoren ist unerlässlich für eine optimale Behandlung und die Verbesserung von Prognosen betroffener Menschen. Den Goldstandard für Gewebeuntersuchungen stellt bis heute die Histopathologie dar, wobei schmerzhafte Patientenbiopsien und komplexe Nachbehandlungen der Gewebeproben notwendig sind. Von der Gewebeentnahme bis zur Diagnosestellung vergeht wertvolle Zeit, in vielen Fällen müssen Biopsien mehrfach wiederholt werden, bis krankhaftes Gewebe vollständig identifiziert und lokalisiert ist. Aus diesem Grund wurden in den letzten Jahrzehnten neuartige optische Technologien entwickelt und angewendet, um Gewebeanalysen zu optimieren und eine patientenfreundlichere Behandlung zu ermöglichen. Nichtlineare, multimodale In-vivo-Bildgebung mit CARS (coherent anti-Stokes Raman scattering), SHG (second harmonic generation) und TPEF (two-photon excited auto-fluorescence) kann hier einen attraktiven Lösungsansatz bieten, welcher nichtinvasiv ist und die Detektion von mikroskopischen Gewebeveränderungen in Echtzeit ermöglicht. Die Implementierung nichtlinearer Bildgebungsverfahren in ein endoskopisches Design für schwer zugängliche Gewebestellen stellt jedoch immer noch eine technologische Herausforderung dar. Die vorliegende Forschungsarbeit präsentiert einen faserbasierten Ansatz für eine multimodale, endoskopische Sonde, wobei eine Multikernfaser für die Führung der Anregungslaser genutzt wird und speziell angefertigte GRIN-Linsen die Strahlformung im Sondenkopf übernehmen. Das Sondendesign kommt vollständig ohne bewegliche Teile oder den Einsatz von elektrischem Strom aus, so dass eine robuste und kompakte Bauform erreicht wird. Die Funktionalität der Sonde wird mit multimodalen Aufnahmen an Dünnschnitten von menschlichem Hautgewebe sowie an frischen Gewebeproben eines Hausschweins demonstriert. Anhand der Ergebnisse kann gezeigt werden, dass multimodale Aufnahmen mit der vorgestellten Sonde qualitativ vergleichbar sind mit konventionellen LSM-Aufnahmen, darüber hinaus ist eine schnellere Bildgebung möglich. Somit hat das präsentierte Sondendesign ein hohes Potential, zukünftig bei klinischen Routineuntersuchungen eingesetzt zu werden
Advanced single molecule localization microscopy for imaging cellular nuclei
No full textIn this PhD research, single molecule localization microscopy (SMLM) was used to image nuclear structures with a resolution down to several nanometers.The scope of this PhD research is to develop a 3D SMLM microscope which can overcome several principle limitations in imaging nuclei in 3D. The advanced improvements during this PhD research include a broad range of research subjects associated to SMLM techniques. Firstly, one of the most common problems of a super-resolution microscope is sample drift, because a small sample drift may result in artefacts and can hamper the resolution. A speckle-based method was developed to correct sample drift without changing the standard design of the SMLM setup. This drift correction method can achieve a resolution of several nanometers. Secondly, another principle problem is that commonly used organic fluorophores are restricted in their photon budget. It is often observed that the chemical structure of fluorophores change after high laser irradiance resulting in photobleaching. A patterned illumination technique was developed which allows the user to define arbitrary regions of interest for illumination with a flat-top intensity profile. Thirdly, for SMLM in particular, a carefully adjusted chemical environment in the sample is recommended to induce sufficiently blinking signals of the organic fluorophores in combination with an appropriate laser irradiance. However, such an imaging buffer can degrade over time and may not be suitable for long time imaging. Nanographene was presented as a new class of fluorophores which have blinking properties without an imaging buffer. Therefore, the nanographenes facilitate a wide range of SMLM applications including bio-imaging and material science. These advanced developments are not only for imaging nuclei, but also applicable to applications in other biological researches and in material science
High-resolution direct stochastic optical reconstruction microscopy of the human kinetochore chromatin
No full textThe kinetochore is a chromatin-protein complex on the chromosome centromere. Kinetochore attache chromosomes to the microtubules of the spindle apparatus, recognize attachment to microtubules, and regulate the progress of mitosis. The kinetochore protein complex is built on the periphery centromere chromatin. It seems that kinetochores react to forces of the spindle apparatus microtubules in their interior. Due to the limited resolving power of the optical microscope and limitations of electron microscopy, the spatial arrangement of kinetochore protein and chromatin complex is unresolved. In this thesis, the human periphery centromere chromatin assembled with CENP-A (kinetochore chromatin in further text), on which kinetochore complex is built, was imaged by high-resolution optical microscopy called direct Stochastic Optical Reconstruction Microscopy (dSTORM). Resolving power of less than 30 nm was achieved, and the architecture of more than 900 kinetochores in different sub-stages of interphase and mitosis was resolved. Kinetochore chromatin was formed in a rectangle between 250 and 400 nm long and between 150 and 270 nm wide. It was composed of parallel and orthogonal lines between 12 and 75 nm wide. The arrangement of kinetochore chromatin in mitosis was narrower and longer than in interphase. In interphase, subtle changes in the dimensions of kinetochore lines were measured. The mitotic toxin nocodazole disturbed kinetochore chromatin organization. The discovered change of arrangement of the kinetochore chromatin assembled with CENP-A during the cell cycle could be the physical mechanism of recognition of proper attachment and positioning of the chromosomes at the equatorial plate. Based on the discovered kinetochore structure and behavior in the cell cycle, a new shoo-lace model of assembly and function of kinetochore chromatin is suggested
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