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The Netherlands Cancer Institute (NKI), Division of Cell Biology, Amsterdam, The Netherlands

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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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  • Practical Guide for Excellent GSDIM Super-Resolution Images

    Do you know that most protists and bacteria lack in one feature that each of our body cell has? Our cells are touch and communicate with one another. They send and receive a variety of signals that coordinate their behavior to act together as a functional multicellular organism. Exploring the way of cellular communication and the ways how the cell surface interacts to organize tissues and body structures is of great interest. Kees Jalink and his team of scientists at the Netherlands Cancer Institute (NKI) in Amsterdam obtained new scientific insights into the molecular architecture of hemidesmosomes, cytoskeletal components, cell surface receptors and vesicular proteins with the help of Ground-State-Depletion (GSD)/ dSTORM microscopy. In this interview, Kees Jalink comments on their developments in imaging chambers, buffer conditions and image analysis to get the perfect super resolution image.
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  • "The Leica Digital Light Sheet Module – a Clever Example of Thinking Out of the Box"

    Bram van den Broek is a postdoctoral fellow at the Netherlands cancer institute in Amsterdam where he supports the advanced microscopy techniques in the laboratory of Kees Jalink. Working with Leica Microsystems as a collaboration partner for beta-testing of microscopes he enjoys very much.
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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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  • Abstracts of the 5th European Super-Resolution User-Club Meeting

    The 5th Super-Resolution User Club Meeting was held in collaboration with Professor Kees Jalink and The Netherlands Cancer Institute (NKI) in Amsterdam. Having the meeting at a location where super-resolution microscopy is used on a daily basis makes a big difference, offering participants the chance to use live cells for workshops and see systems working in their true environments. Thanks also to the scientists that supported the meeting by coming and giving talks. As super-resolution continues to grow in importance in research, we recognize the need to come together to network, share information and experiences. Here we present the abstracts of the talks.
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  • Video Interviews with Kees Jalink

    Kees Jalink's group at the Netherlands Cancer Institute in Amsterdam, The Netherlands, explores signal transduction pathways and cell adhesion processes in cancer cells. In his eyes especially the new three-dimensional nanoscopic view of the relevant structure of interest is an essential feature to get the full picture.
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  • Abstracts of the 4th European Super-Resolution User-Club Meeting

    The 4th Super-Resolution User Club Meeting was held in collaboration with Christian Eggeling and the Weatherall Institute of Molecular Medicine in Oxford, UK. Here we present the abstracts of the talks and interviews with participants.
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  • A mTurquoise-Based cAMP Sensor for Both FLIM and Ratiometric Read-Out Has Improved Dynamic Range

    FRET-based sensors for cyclic Adenosine Mono Phosphate (cAMP) have revolutionized the way in which this important intracellular messenger is studied. The currently prevailing sensors consist of the cAMP-binding protein Epac1, sandwiched between suitable donor- and acceptor fluorescent proteins (FPs).
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  • The First Supercontinuum Confocal that Adapts to the Sample

    Until now, biological and medical research fluorescence imaging in multi-user facilities or institutes has been limited by the type or number of dyes that could be excited. The Leica TCS SP5 X supercontinuum confocal unites the broadband capabilities of the Leica TCS SP5 AOBS® and the freedom and flexibility to select any excitation line within the continuous range of 470 to 670 nm.
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  • A Comparison of Donor-Acceptor Pairs for Genetically Encoded FRET Sensors: Application to the Epac cAMP Sensor as an Example

    We recently reported on CFP-Epac-YFP, an Epac-based single polypeptide FRET reporter to resolve cAMP levels in living cells. In this study, we compared and optimized the fluorescent protein donor/acceptor pairs for use in biosensors such as CFP-Epac-YFP. Our strategy was to prepare a wide range of constructs consisting of different donor and acceptor fluorescent proteins separated by a short linker.
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  • Correcting Confocal Acquisition to Optimize Imaging of Fluorescence Resonance Energy Transfer by Sensitized Emission

    Imaging of fluorescence resonance energy transfer (FRET) between suitable fluorophores is increasingly being used to study cellular processes with high spatiotemporal resolution. The genetically encoded Cyan (CFP) and Yellow (YFP) variants of Green Fluorescent Protein have become the most popular donor and acceptor pair in cell biology. FRET between these fluorophores can be imaged by detecting sensitized emission. This technique, for which CFP is excited and transfer is detected as emission of YFP, is sensitive, fast, and straightforward, provided that proper corrections are made. In this study, the detection of sensitized emission between CFP and YFP by confocal microscopy is optimized.
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