TY - THES A1 - Kurz, Andreas T1 - Correlative live and fixed cell superresolution microscopy T1 - Korrelative hochauflösende Mikroskopie an lebenden und fixierten Zellen N2 - Over the last decade life sciences have made an enormous leap forward. The development of complex analytical instruments, in particular in fluorescence microscopy, has played a decisive role in this. Scientist can now rely on a wide range of imaging techniques that offer different advantages in terms of optical resolution, recording speed or living cell compatibility. With the help of these modern microscopy techniques, multi-protein complexes can be resolved, membrane receptors can be counted, cellular pathways analysed or the internalisation of receptors can be tracked. However, there is currently no universal technique for comprehensive experiment execution that includes dynamic process capture and super resolution imaging on the same target object. In this work, I built a microscope that combines two complementary imaging techniques and enables correlative experiments in living and fixed cells. With an image scanning based laser spot confocal microscope, fast dynamics in several colors with low photodamage of the cells can be recorded. This novel system also has an improved resolution of 170 nm and was thoroughly characterized in this work. The complementary technique is based on single molecule localization microscopy, which can achieve a structural resolution down to 20-30 nm. Furthermore I implemented a microfluidic pump that allows direct interaction with the sample placed on the microscope. Numerous processes such as living cell staining, living cell fixation, immunostaining and buffer exchange can be observed and performed directly on the same cell. Thus, dynamic processes of a cell can be frozen and the structures of interest can be stained and analysed with high-resolution microscopy. Furthermore, I have equipped the detection path of the single molecule technique with an adaptive optical element. With the help of a deformable mirror, imaging functions can be shaped and information on the 3D position of the individual molecules can be extracted. N2 - Im letzten Jahrzehnt hat der Bereich der Lebenswissenschaften einen enormen Sprung nach vorne gemacht. Maßgeblich dafür waren die Entwicklung von komplexen Analysegeräten insbesondere in der Fluoreszenz Mikroskopie. Die Anwender können nun auf eine Vielzahl von Bildgebungstechniken zurückgreifen die unterschiedliche Vorzüge hinsichtlich optischer Auflösung, Aufnahmegeschwindigkeit oder Lebend Zell Kompatibilität bieten. Mithilfe dieser modernen Mikroskopietechniken lassen sich beispielsweise Multiproteinkomplexe auflösen, Membranrezeptoren zählen, zelluläre Signalwege analysieren oder die Internalisierung von Rezeptoren verfolgen. Für eine umfassende Experimentdurchführung, die Erfassung dynamischer Prozesse sowie superhochauflösende Bildgebung an ein und demselben Zielobjekt beinhalten, gibt es derzeit keine einheitliche Technik. In dieser Arbeit habe ich ein Mikroskop aufgebaut, das zwei komplementäre Bildgebungstechniken vereint und korrelative Experimente von lebend zu fixierten Zellen ermöglicht. Mit einem Image Scanning basierten Konfokal Mikroskop können schnelle Dynamiken in mehreren Farben mit geringer Photoschädigung der Zellen aufgenommen werden. Dieses neuartige System weist zudem eine Auflösungsverbesserung von 170 nm auf und wurde im Rahmen der Arbeit ausführlich charakterisiert. Die komplementäre Technik basiert auf der Einzel-Molekül Lokalisations Mikroskopie, mit der sich eine strukturelle Auflösung von bis zu 20 nm erreichen lässt. Desweiteren habe ich eine Mikrofluidpumpe implementiert, die eine direkte Interaktion mit der auf dem Mikroskop platzierten Probe erlaubt. Zahlreiche Prozesse wie Lebend-Zell Färbung, Lebend-Zell Fixierung, Immuno-Färbung und Puffertausch können damit direkt an der gleichen Zelle beobachtet und durchgeführt werden. So können dynamische Prozesse einer Zelle sozusagen eingefroren werden und die Strukturen von Interesse gefärbt und mit höchstauflösender Mikroskopie analysiert werden. Desweiteren habe ich den Detektionspfad der Einzel-Molekül Technik mit einem adaptiven optischen Element ausgestattet. Mithilfe eines deformierbaren Spiegels lässt sich so Abbildungsfunktion formen und Information zur 3D Position der einzelnen Moleküle gewinnen. KW - Einzelmolekülmikroskopie KW - Adaptive Optik KW - Image-Scanning Microscope KW - Correlative microscopy KW - Adaptive Optics Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-199455 ER - TY - JOUR A1 - Berberich, Andreas A1 - Kurz, Andreas A1 - Reinhard, Sebastian A1 - Paul, Torsten Johann A1 - Burd, Paul Ray A1 - Sauer, Markus A1 - Kollmannsberger, Philip T1 - Fourier Ring Correlation and anisotropic kernel density estimation improve deep learning based SMLM reconstruction of microtubules JF - Frontiers in Bioinformatics N2 - Single-molecule super-resolution microscopy (SMLM) techniques like dSTORM can reveal biological structures down to the nanometer scale. The achievable resolution is not only defined by the localization precision of individual fluorescent molecules, but also by their density, which becomes a limiting factor e.g., in expansion microscopy. Artificial deep neural networks can learn to reconstruct dense super-resolved structures such as microtubules from a sparse, noisy set of data points. This approach requires a robust method to assess the quality of a predicted density image and to quantitatively compare it to a ground truth image. Such a quality measure needs to be differentiable to be applied as loss function in deep learning. We developed a new trainable quality measure based on Fourier Ring Correlation (FRC) and used it to train deep neural networks to map a small number of sampling points to an underlying density. Smooth ground truth images of microtubules were generated from localization coordinates using an anisotropic Gaussian kernel density estimator. We show that the FRC criterion ideally complements the existing state-of-the-art multiscale structural similarity index, since both are interpretable and there is no trade-off between them during optimization. The TensorFlow implementation of our FRC metric can easily be integrated into existing deep learning workflows. KW - dSTORM KW - deep learning–artificial neural network (DL-ANN) KW - single molecule localization microscopy KW - microtubule cytoskeleton KW - super-resolution Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-261686 VL - 1 ER - TY - JOUR A1 - Derakhshani, Shaghayegh A1 - Kurz, Andreas A1 - Japtok, Lukasz A1 - Schumacher, Fabian A1 - Pilgram, Lisa A1 - Steinke, Maria A1 - Kleuser, Burkhard A1 - Sauer, Markus A1 - Schneider-Schaulies, Sibylle A1 - Avota, Elita T1 - Measles virus infection fosters dendritic cell motility in a 3D environment to enhance transmission to target cells in the respiratory epithelium JF - Frontiers in Immunology N2 - Transmission of measles virus (MV) from dendritic to airway epithelial cells is considered as crucial to viral spread late in infection. Therefore, pathways and effectors governing this process are promising targets for intervention. To identify these, we established a 3D respiratory tract model where MV transmission by infected dendritic cells (DCs) relied on the presence of nectin-4 on H358 lung epithelial cells. Access to recipient cells is an important prerequisite for transmission, and we therefore analyzed migration of MV-exposed DC cultures within the model. Surprisingly, enhanced motility toward the epithelial layer was observed for MV-infected DCs as compared to their uninfected siblings. This occurred independently of factors released from H358 cells indicating that MV infection triggered cytoskeletal remodeling associated with DC polarization enforced velocity. Accordingly, the latter was also observed for MV-infected DCs in collagen matrices and was particularly sensitive to ROCK inhibition indicating infected DCs preferentially employed the amoeboid migration mode. This was also implicated by loss of podosomes and reduced filopodial activity both of which were retained in MV-exposed uninfected DCs. Evidently, sphingosine kinase (SphK) and sphingosine-1-phosphate (S1P) as produced in response to virus-infection in DCs contributed to enhanced velocity because this was abrogated upon inhibition of sphingosine kinase activity. These findings indicate that MV infection promotes a push-and-squeeze fast amoeboid migration mode via the SphK/S1P system characterized by loss of filopodia and podosome dissolution. Consequently, this enables rapid trafficking of virus toward epithelial cells during viral exit. KW - dendritic cell KW - cell migration KW - measles virus KW - 3D tissue model KW - sphingosine-1-phosphate Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201818 VL - 10 IS - 1294 ER -