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  • The Molecular Architecture of Hemidesmosomes as Revealed by Super-Resolution Microscopy

    Hemidesmosomes have been extensively studied by immunofluorescence microscopy, but due to its limited resolution, their precise organization remained poorly understood. We studied hemidesmosome organization in cultured keratinocytes by 2- and 3-color super-resolution microscopy. We observed that in the cell periphery, nascent hemidesmosomes are associated with individual keratin filaments and that β4 is distributed along rather than under keratin filaments. By applying innovative methods to quantify molecular distances, we demonstrate that the hemidesmosomal plaque protein plectin interacts simultaneously and asymmetrically with β4 and keratin.
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  • TIRF Publication List

    This monthly updated references list presents current papers using Leica AM TIRF in the major application fields for TIRF microscopy.
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  • Co-Orientation: Quantifying Simultaneous Co-Localization and Orientational Alignment of Filaments in Light Microscopy

    Co-localization analysis is a widely used tool to seek evidence for functional interactions between molecules in different color channels in microscopic images. Here we extend the basic co-localization analysis by including the orientations of the structures on which the molecules reside. We refer to the combination of co-localization of molecules and orientational alignment of the structures on which they reside as co-orientation. Because the orientation varies with the length scale at which it is evaluated, we consider this scale as a separate informative dimension in the analysis. Additionally we introduce a data driven method for testing the statistical significance of the co-orientation and provide a method for visualizing the local co-orientation strength in images. We demonstrate our methods on simulated localization microscopy data of filamentous structures, as well as experimental images of similar structures acquired with localization microscopy in different color channels.
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  • Video Talk by Daniel Axelrod: Total Internal Reflection Fluorescence (TIRF) Microscopy

    Total Internal Reflection Fluorescence (TIRF) Microscopy is a technique that only illuminates dye molecules near a surface. In this video, the pioneer of TIRF Microscopy describes what this technique is used for, explains the principles of the evanescent wave, gives many examples of different microscope configurations used in TIRF, and shows how polarized light TIRF can be used to image membrane orientation.
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  • Universal PAINT – Dynamic Super-Resolution Microscopy

    Super-resolution microscopy techniques have revolutionized biology for the last ten years. With their help cellular components can now be visualized at the size of a protein. Nevertheless, imaging living cells is a challenge for most of the super-resolution principles.
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  • Video Interview with William Hughes

    William Hughes works at the Garvan Institute of Medical Research, Sydney (Australia). In his Lab Head position he is interested in the causes of diabetes particularly looking at changes in exocytic behavior of pancreatic beta cells as well as fat and muscle cells. TIRF microscopy is predestined for researchers looking at cellular processes near the cytoplasmic membrane.
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  • Live-Cell Imaging Evolves to Find New Niches

    Since its introduction in the 1600s, improvements in microscope technology have continually broadened the types of cells and cellular processes that can be studied. Advances in automation have made this already-simple tool faster and more capable, and time-lapse imaging reveals function and dynamics in addition to structure. Live-cell imaging has enabled us to witness incredible moments in biology in unprecedented detail. Even embryogenesis – the process of cell division and cellular differentiation that occurs at the earliest stages of life – has recently been captured.
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  • Webinar: From Epifluorescence to Super-Resolution in 3D

    This webinar will illustrate results obtained by biochemical, Epifluorescence, TIRFM, Confocal and GSD techniques. Depending on the aim of experimental question, different imaging techniques deliver insights into varying aspects of intracellular pathways. To achieve "True-to-detail imaging" of the spatial arrangement of proteins and other biomolecules in cells, GSDIM achieves resolutions up to 20 nm in x and y direction – beyond the diffraction limit of light microscopy. But Super-resolution microscopy can be applied in the axial (z-) direction, too. A recent commercial implementation of the astigmatism approach will be discussed in more detail during this webinar.
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  • Webinar: Challenges in Live Cell Imaging

    Live cell imaging is one tool in the cell biologist's tool box. There are, however, a variety of technologies and methods to achieve this. In this webinar, three practicing researchers discuss: Their use of live cell imaging and how it has helped them achieve publishable results.
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  • Controlling the TIRF Penetration Depth is Mandatory for Reproducible Results

    The main feature of total internal reflection fluorescence (TIRF) microscopy is the employment of an evanescent wave for the excitation of fluorophores instead of using direct light. A property of the evanescent wave, which arises from the glass/water or glass/specimen interface, is that its propagation in z-direction gradually degrades, limiting its penetration depth into the specimen to some hundred nanometers.
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  • Protein Transport Processes at the Apical Membrane of Polarized Epithelial Cells

    Due to their special role in organ function and the exchange of biological components some body cells developed certain polarization characteristics. These are reflected in differences of their plasma membrane composition. The essential and fascinating task of polarized protein transport in epithelial cells is to get the right protein into the right membrane.
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  • Total Internal Reflection Fluorescence (TIRF) Microscopy

    Total internal reflection fluorescence (TIRF) is a special technique in fluorescence microscopy developed by Daniel Axelrod at the University of Michigan, Ann Arbor in the early 1980s. TIRF microscopy delivers images with an outstandingly high axial resolution below 100 nm. This allows the observation of membrane-associated processes.
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  • Applications of TIRF Microscopy in Life Science Research

    The special feature of TIRF microscopy is the employment of an evanescent field for fluorophore excitation. Unlike standard widefield fluorescence illumination procedures with arc lamps, LEDs or lasers, the evanescent field only penetrates the specimen by about 100 nm starting from the coverslip/medium interface.
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  • Live-cell Imaging Techniques

    The understanding of complex and/or fast cellular dynamics is an important step for exploring biological processes. Therefore, today’s life science research is increasingly focusing on dynamic processes like cell migration, morphological changes of cells, organs or whole animals and physiological (e.g. changes of intracellular ion composition) events in living specimens in real time.
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  • TIRF Microscopy of the Apical Membrane of Polarized Epithelial Cells

    Application of TIRF microscopy (Total Internal Reflection Fluorescence) allows the visualization of structures at the apical surface of polarized epithelial cells that have been hidden in conventional fluorescence microscopy images. Hence, the approach reveals new insights into the composition of this characteristic cell pole that elucidate processes in apical protein trafficking.
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  • A Guide to Super-Resolution Fluorescence Microscopy

    For centuries, cell biology has been based on light microscopy and at the same time been limited by its optical resolution. However, several new technologies have been developed recently that bypass this limit.
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  • Exploring Cell Logistics

    Using TIRF microscopy, scientists have been able to take a closer look at intracellular transport processes with the example of the galactose-binding protein Galectin-3, which has been identified as a potential apical sorting receptor.
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