TY - JOUR A1 - Nanguneri, Siddharth A1 - Flottmann, Benjamin A1 - Horstmann, Heinz A1 - Heilemann, Mike A1 - Kuner, Thomas T1 - Three-Dimensional, Tomographic Super-Resolution Fluorescence Imaging of Serially Sectioned Thick Samples JF - PLoS One N2 - Three-dimensional fluorescence imaging of thick tissue samples with near-molecular resolution remains a fundamental challenge in the life sciences. To tackle this, we developed tomoSTORM, an approach combining single-molecule localization-based super-resolution microscopy with array tomography of structurally intact brain tissue. Consecutive sections organized in a ribbon were serially imaged with a lateral resolution of 28 nm and an axial resolution of 40 nm in tissue volumes of up to 50 \(\mu\)mx50\(\mu\)mx2.5\(\mu\)m. Using targeted expression of membrane bound (m)GFP and immunohistochemistry at the calyx of Held, a model synapse for central glutamatergic neurotransmission, we delineated the course of the membrane and fine-structure of mitochondria. This method allows multiplexed super-resolution imaging in large tissue volumes with a resolution three orders of magnitude better than confocal microscopy. KW - architecture KW - rat calyx KW - in-vivo KW - microscopy KW - resolution KW - proteins KW - transmission KW - ultrastructure KW - reconstruction KW - localization Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-134434 VL - 7 IS - 5 ER - TY - JOUR A1 - Muranyi, Walter A1 - Malkusch, Sebastian A1 - Müller, Barbara A1 - Heilemann, Mike A1 - Kräusslich, Hans-Georg T1 - Super-Resolution Microscopy Reveals Specific Recruitment of HIV-1 Envelope Proteins to Viral Assembly Sites Dependent on the Envelope C-Terminal Tail JF - PLoS Pathogens N2 - The inner structural Gag proteins and the envelope (Env) glycoproteins of human immunodeficiency virus (HIV-1) traffic independently to the plasma membrane, where they assemble the nascent virion. HIV-1 carries a relatively low number of glycoproteins in its membrane, and the mechanism of Env recruitment and virus incorporation is incompletely understood. We employed dual-color super-resolution microscopy visualizing Gag assembly sites and HIV-1 Env proteins in virus-producing and in Env expressing cells. Distinctive HIV-1 Gag assembly sites were readily detected and were associated with Env clusters that always extended beyond the actual Gag assembly site and often showed enrichment at the periphery and surrounding the assembly site. Formation of these Env clusters depended on the presence of other HIV-1 proteins and on the long cytoplasmic tail (CT) of Env. CT deletion, a matrix mutation affecting Env incorporation or Env expression in the absence of other HIV-1 proteins led to much smaller Env clusters, which were not enriched at viral assembly sites. These results show that Env is recruited to HIV-1 assembly sites in a CT-dependent manner, while Env\((\Delta CT)\) appears to be randomly incorporated. The observed Env accumulation surrounding Gag assemblies, with a lower density on the actual bud, could facilitate viral spread in vivo. Keeping Env molecules on the nascent virus low may be important for escape from the humoral immune response, while cell-cell contacts mediated by surrounding Env molecules could promote HIV-1 transmission through the virological synapse. KW - ENV KW - fluorescent-probes KW - type-1 matrix KW - glycoprotein incorporation KW - GP41 cytoplasmic tail KW - human immunodeficiency virus KW - cellular proteins KW - plasma membrane KW - virions KW - particles Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-131235 VL - 9 IS - 2 ER - TY - JOUR A1 - Dietz, Mariana S. A1 - Hasse, Daniel A1 - Ferraris, Davide M. A1 - Göhler, Antonia A1 - Niemann, Hartmut H. A1 - Heilemann, Mike T1 - Single-molecule photobleaching reveals increased MET receptor dimerization upon ligand binding in intact cells JF - BMC Biophysics N2 - Background: The human receptor tyrosine kinase MET and its ligand hepatocyte growth factor/scatter factor are essential during embryonic development and play an important role during cancer metastasis and tissue regeneration. In addition, it was found that MET is also relevant for infectious diseases and is the target of different bacteria, amongst them Listeria monocytogenes that induces bacterial uptake through the surface protein internalin B. Binding of ligand to the MET receptor is proposed to lead to receptor dimerization. However, it is also discussed whether preformed MET dimers exist on the cell membrane. Results: To address these issues we used single-molecule fluorescence microscopy techniques. Our photobleaching experiments show that MET exists in dimers on the membrane of cells in the absence of ligand and that the proportion of MET dimers increases significantly upon ligand binding. Conclusions: Our results indicate that partially preformed MET dimers may play a role in ligand binding or MET signaling. The addition of the bacterial ligand internalin B leads to an increase of MET dimers which is in agreement with the model of ligand-induced dimerization of receptor tyrosine kinases. KW - single-molecule photobleaching KW - fluorescence correlation spectroscopy KW - fluorescence KW - EGF receptor KW - rat hepatocytes KW - structural insights KW - Scatter factor KW - SEMA domain KW - hepatocyte-growth-factor KW - invasion protein-INLB KW - listeria-monocytogenes KW - tyrosine kinase KW - living cells KW - dimerization KW - MET receptor KW - Signal transduction Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-121835 SN - 2046-1682 VL - 6 IS - 6 ER - TY - JOUR A1 - Sporbert, Anje A1 - Cseresnyes, Zoltan A1 - Heidbreder, Meike A1 - Domaing, Petra A1 - Hauser, Stefan A1 - Kaltschmidt, Barbara A1 - Kaltschmidt, Christian A1 - Heilemann, Mike A1 - Widera, Darius T1 - Simple Method for Sub-Diffraction Resolution Imaging of Cellular Structures on Standard Confocal Microscopes by Three-Photon Absorption of Quantum Dots JF - PLoS ONE N2 - This study describes a simple technique that improves a recently developed 3D sub-diffraction imaging method based on three-photon absorption of commercially available quantum dots. The method combines imaging of biological samples via tri-exciton generation in quantum dots with deconvolution and spectral multiplexing, resulting in a novel approach for multi-color imaging of even thick biological samples at a 1.4 to 1.9-fold better spatial resolution. This approach is realized on a conventional confocal microscope equipped with standard continuous-wave lasers. We demonstrate the potential of multi-color tri-exciton imaging of quantum dots combined with deconvolution on viral vesicles in lentivirally transduced cells as well as intermediate filaments in three-dimensional clusters of mouse-derived neural stem cells (neurospheres) and dense microtubuli arrays in myotubes formed by stacks of differentiated C2C12 myoblasts. KW - HIV KW - stem-cell KW - infection Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130963 VL - 8 IS - 5 ER - TY - JOUR A1 - Lando, David A1 - Endesfelder, Ulrike A1 - Berger, Harald A1 - Subramanian, Lakxmi A1 - Dunne, Paul D. A1 - McColl, James A1 - Klenerman, David A1 - Carr, Antony M. A1 - Sauer, Markus A1 - Allshire, Robin C. A1 - Heilemann, Mike A1 - Laue, Ernest D. T1 - Quantitative single-molecule microscopy reveals that CENP-A\(^{Cnp1}\) deposition occurs during G2 in fission yeast JF - Open Biology N2 - The inheritance of the histone H3 variant CENP-A in nucleosomes at centromeres following DNA replication is mediated by an epigenetic mechanism. To understand the process of epigenetic inheritance, or propagation of histones and histone variants, as nucleosomes are disassembled and reassembled in living eukaryotic cells, we have explored the feasibility of exploiting photo-activated localization microscopy (PALM). PALM of single molecules in living cells has the potential to reveal new concepts in cell biology, providing insights into stochastic variation in cellular states. However, thus far, its use has been limited to studies in bacteria or to processes occurring near the surface of eukaryotic cells. With PALM, one literally observes and 'counts' individual molecules in cells one-by-one and this allows the recording of images with a resolution higher than that determined by the diffraction of light (the so-called super-resolution microscopy). Here, we investigate the use of different fluorophores and develop procedures to count the centromere-specific histone H3 variant CENP-A\(^{Cnp1}\) with single-molecule sensitivity in fission yeast (Schizosaccharomyces pombe). The results obtained are validated by and compared with ChIP-seq analyses. Using this approach, CENP-A\(^{Cnp1}\) levels at fission yeast (S. pombe) centromeres were followed as they change during the cell cycle. Our measurements show that CENP-A(Cnp1) is deposited solely during the G2 phase of the cell cycle. KW - nucleosome KW - fission yeast KW - identification KW - propagation KW - CSE4, CENP-A KW - CENP-A KW - schizosaccaromyces-pombe KW - fluorescent protein KW - centomeres KW - superresolution KW - chromatin KW - centromere KW - ingle-molecule microscopy Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-134682 VL - 2 IS - 120078 ER - TY - JOUR A1 - Endesfelder, Ulrike A1 - Malkusch, Sebastian A1 - Flottmann, Benjamin A1 - Mondry, Justine A1 - Liguzinski, Piotr A1 - Verveer, Peter J. A1 - Heilemann, Mike T1 - Chemically Induced Photoswitching of Fluorescent Probes - A General Concept for Super-Resolution Microscopy N2 - We review fluorescent probes that can be photoswitched or photoactivated and are suited for single-molecule localization based super-resolution microscopy. We exploit the underlying photochemical mechanisms that allow photoswitching of many synthetic organic fluorophores in the presence of reducing agents, and study the impact of these on the photoswitching properties of various photoactivatable or photoconvertible fluorescent proteins. We have identified mEos2 as a fluorescent protein that exhibits reversible photoswitching under various imaging buffer conditions and present strategies to characterize reversible photoswitching. Finally, we discuss opportunities to combine fluorescent proteins with organic fluorophores for dual-color photoswitching microscopy. KW - Super-Resolution Microscopy KW - photoswitchable organic fluorophores KW - fluorescent proteins KW - super-resolution KW - PALM KW - dSTORM Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-74896 ER - TY - JOUR A1 - Endesfelder, Ulrike A1 - Malkusch, Sebastian A1 - Flottmann, Benjamin A1 - Mondry, Justine A1 - Liguzinski, Piotr A1 - Verveer, Peter J. A1 - Heilemann, Mike T1 - Chemically Induced Photoswitching of Fluorescent Probes - A General Concept for Super-Resolution Microscopy JF - Molecules N2 - We review fluorescent probes that can be photoswitched or photoactivated and are suited for single-molecule localization based super-resolution microscopy. We exploit the underlying photochemical mechanisms that allow photoswitching of many synthetic organic fluorophores in the presence of reducing agents, and study the impact of these on the photoswitching properties of various photoactivatable or photoconvertible fluorescent proteins. We have identified mEos2 as a fluorescent protein that exhibits reversible photoswitching under various imaging buffer conditions and present strategies to characterize reversible photoswitching. Finally, we discuss opportunities to combine fluorescent proteins with organic fluorophores for dual-color photoswitching microscopy. KW - Photoactivated localization microscopy KW - Fusion proteins KW - Molecules KW - Patterns KW - Switch KW - Limit KW - Time KW - photoswitchable organic fluorophores KW - fluorescent proteins KW - super-resolution KW - PALM KW - dSTORM Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-134080 VL - 16 IS - 4 ER - TY - JOUR A1 - Eckhardt, Manon A1 - Anders, Maria A1 - Muranyi, Walter A1 - Heilemann, Mike A1 - Krijnse-Locker, Jacomine A1 - Müller, Barbara T1 - A SNAP-Tagged Derivative of HIV-1-A Versatile Tool to Study Virus-Cell Interactions JF - PLoS ONE N2 - Fluorescently labeled human immunodeficiency virus (HIV) derivatives, combined with the use of advanced fluorescence microscopy techniques, allow the direct visualization of dynamic events and individual steps in the viral life cycle. HIV proteins tagged with fluorescent proteins (FPs) have been successfully used for live-cell imaging analyses of HIV-cell interactions. However, FPs display limitations with respect to their physicochemical properties, and their maturation kinetics. Furthermore, several independent FP-tagged constructs have to be cloned and characterized in order to obtain spectral variations suitable for multi-color imaging setups. In contrast, the so-called SNAP-tag represents a genetically encoded non-fluorescent tag which mediates specific covalent coupling to fluorescent substrate molecules in a self-labeling reaction. Fusion of the SNAP-tag to the protein of interest allows specific labeling of the fusion protein with a variety of synthetic dyes, thereby offering enhanced flexibility for fluorescence imaging approaches. Here we describe the construction and characterization of the HIV derivative HIV(SNAP), which carries the SNAP-tag as an additional domain within the viral structural polyprotein Gag. Introduction of the tag close to the C-terminus of the matrix domain of Gag did not interfere with particle assembly, release or proteolytic virus maturation. The modified virions were infectious and could be propagated in tissue culture, albeit with reduced replication capacity. Insertion of the SNAP domain within Gag allowed specific staining of the viral polyprotein in the context of virus producing cells using a SNAP reactive dye as well as the visualization of individual virions and viral budding sites by stochastic optical reconstruction microscopy. Thus, HIV(SNAP) represents a versatile tool which expands the possibilities for the analysis of HIV-cell interactions using live cell imaging and sub-diffraction fluorescence microscopy. KW - Human-immunodeficiency-virus KW - Fusion proteins KW - Live cells KW - Fluorescence microscopy KW - Stimulated-emission KW - Plasma-membrane KW - Living cells KW - Real-time KW - TYPE-1 KW - GAG Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-133534 VL - 6 IS - 7 ER -