Multidimensional super-resolution imaging of membrane receptors

Project Description
dSTORM imaging of the cytoskeletal network of mammalian cells (COS-7 cells) with five different Alexa Fluor and ATTO dyes spanning the visible wavelength range. Wide-field immunofluorescence images (TIRF microscopy) of microtubules are shown on the left side. Experiments were performed in PBS, pH 7.4, 10–100 mM MEA, with a frame rate of 10–20 Hz and excitation intensities of 1–4 kW cm-2. Since living cells contain glutathione at up to 10 mM concentrations live-cell dSTORM is likewise feasible (III, 16).

Membrane receptor-activated signal transduction pathways are integral to cellular functions and disease mechanisms in humans. However, until now two obstacles impede the exploitation of quantitative data about the dynamic architecture of membrane receptors: selective and efficient labeling of membrane receptors and the resolution limit of classical optical microscopy. Such quantitative data is of special importance considering the existence of confined plasma membrane compartments, i.e. nanodomains or clusters with a supposed size of 5-300 nm required for subcompartimentalization and associated function. We intend to map the distribution and quantify the spatial organization of membrane receptors in the context of other extra- and intracellular molecules at close to molecular resolution using multidimensional single-molecule based super-resolution imaging methods such as PhotoActivated Localization Microscopy (PALM) and direct Stochastic Optical Reconstruction Microscopy (dSTORM).

We will focus our attention on membrane receptors studied within the framework of the CRC/TR but will include also other membrane and membrane-associated cytoskeletal proteins and lipids to investigate the dynamic organization of extracellular membrane molecules proposed to neighboring cells. Our studies include single isolated adherent cells as well as tissue slices and tumor spheroids using wide-field, total-internal reflection (TIR), and single-plane illumination (SPIM) localization microscopy to decipher the influence of cell-cell interactions on the membrane protein network within a realistic setting. In order to achieve this ambitious goal and quantify the spatial distribution of more than 10 different target molecules in a single experiment, we aim to develop a new method for high-throughput protein localization and quantification studies that runs cycles of fluorescence tagging and super-resolution imaging using the same fluorophore. In order to remove the fluorophore from the sample before the next round of fluorescence tagging and imaging, we will develop and optimize different approaches including photobleaching, and photocleaving of fluorophore linkers. Alternatively, we intend to use the transient binding of short fluorescently labeled oligonucleotides for multiplexed super-resolution imaging. Here, the molecules of interest will be labeled with a short specific DNA sequence. Upon transient binding of the complementary fluorophore-labeled oligonucleotide the molecules will be localized. Using different DNA sequences for unequivocal labeling of the molecules of interest, cycles of washing, addition of the next unique fluorophore-labeled oligonucleotide, and localization microscopy can be run. In parallel labeling protocols have to be optimized to enable efficient multi-target labeling and imaging with maximal structure preservation. The use of the same fluorophore for all target molecules ensures comparable detection efficiencies and simplifies quantification of localization data. That is, we aim to introduce a powerful method for the quantitative visualization of the entire dynamic toponome map of membrane receptors.

Title Year Authors Journal Links
Imaging cellular ultrastructures using expansion microscopy (U-ExM) 2019 Gambarotto, D., Zwettler, F. U., Le Guennec, M., Schmidt-Cernohorska, M., Fortun, D., Borgers, S., Heine, J., Schloetel, J.-G., Reuss, M., Unser, M., Boyden, E. S., Sauer, M., Hamel, V., and Guichard, P. Nat Methods More
Sharpening emitter localization in front of a tuned mirror 2018 Heil, H., Schreiber, B., Götz, R., Emmerling, M., Dabauvalle, M.-C., Krohne, G., Höfling, S., Kamp, M., Sauer, M. and Heinze, K. G. Light Sci Appl More
Automated distinction of shearing and distortion artefacts in structured illumination microscopy 2018 Förster, R., Müller, W., Richter, R., and Heintzmann, R. Opt Express More
Bioorthogonal click chemistry enables site-specific fluorescence labeling of functional NMDA receptors for super-resolution imaging 2018 Neubert, F., Beliu, G., Terpitz, U., Werner C., Geis, C., Sauer, M., and Doose, S. Angew Chem Int Ed More
Single-molecule localization microscopy in eukaryotes 2017 Sauer, M., and Heilemann, M. Chem Rev More
Noise-corrected principal component analysis of fluorescence lifetime imaging data 2017 Le Marois, A., Labouesse, S., Suhling, K., and Heintzmann, R. J Biophotonics More
Cyanine conformational restraint in the far-red range 2017 Michie, M.S., Götz, R., Franke, C., Bowler, M., Kumari, N., Magidison, V., Levitus, M.L., Loncarek, J., Sauer, M., and Schnermann, M. J Am Chem Soc
Fast structured illumination microscopy using rolling shutter cameras 2016 Song, L. Y., Lu-Walther, H. W., Forster, R., Jost, A., Kielhorn, M., Zhou, J. Y., and Heintzmann, R. Measurement Science and Technology More
Human autoantibodies to amphiphysin induce defective presynaptic vesicle dynamics and composition 2016 Werner, C., Pauli, M., Doose, S., Weishaupt, A., Haselmann, H., Grunewald, B., Sauer, M., Heckmann, M., Toyka, K. V., Asan, E., Sommer, C., and Geis, C. Brain More
Gephyrin-binding peptidesvisualize postsynaptic sites and modulate neurotranmission 2016 Maric, H. M., Hausrat, T. J., Neubert, F., Dalby, N. O., Doose, S., Sauer, M., Kneussel, M., and Stromgaard, K. Nat Chem Biol More
Calibrating photon counts from a single image 2016 Heintzmann, R., Relich, P.K., Nieuwenhuizen, R.P.J., Lidke, K.A., and Rieger, B. arXiv:1611.05654v2 More
Optical photon reassignment with increased axial resolution by structured illumination 2016 Roth, S. and Heintzmann, R. Methods Appl Fluoresc More
Nonlinear Structured Illumination Using a Fluorescent Protein Activating at the Readout Wavelength 2016 Lu-Walther, H.-W., Hou, W., Kielhorn, M., Arai, Y., Nagai, T., Kessels, M. M., Qualmann, B., Heintzmann, R. PLoS ONE More
The CsrA-FliW network controls polar localization of the dual-function flagellin mRNA in Campylobacter jejuni 2016 Dugar, G., Svensson, S. L., Bischler, T., Waldchen, S., Reinhardt, R., Sauer, M., and Sharma, C. M. Nat Commun More
Quantitative super-resolution imaging of Bruchpilot distinguishes active zone states 2014 Ehmann, N., van de Linde, S., Alon, A., Ljaschenko, D., Keung, X. Z., Holm, T., Rings, A., DiAntonio, A., Hallermann, S., Ashery, U., Heckmann, M., Sauer, M. and Kittel, R. J. Nat Commun More
Comprehensive FISH Probe Design Tool Applied to Imaging Human Immunoglobulin Class Switch Recombination 2012 Nedbal, J., Hobson, P.S., Fear, D. J., Heintzmann, R., Gould, H. J. PLoS ONE More
Sub-diffraction imaging on standard microscopes through photobleaching microscopy with nonlinear processing 2012 Munck, S., Miskiewicz, K., Sannerud, R., Menchon, S. A., Jose, L, Heintzmann, R., Verstreken, P. and Annaert, W. J Cell Sci More
Bayesian localization microscopy reveals nanoscale podosome dynamics 2012 Cox, S., Rosten, E., Monypenny, J., Jovanovic-Talisman, T., Burnette, D. T., Lippincott-Schwartz, J., Jones, G. E. and Heintzmann, R. Nat Methods More
Super-resolution imaging visualizes the eightfold symmetry of gp210 proteins around the nuclear pore complex and resolves the central channel with nanometer resolution 2012 Löschberger, A., S. van de Linde, M. C. Dabauvalle, B. Rieger, M. Heilemann, Krohne, G. and Sauer, M. J Cell Sci More
Direct stochastic optical reconstruction microscopy with standard fluorescent probes 2011 van de Linde, S., Löschberger, A., Klein, T., Heidbreder, M., Wolter, S., Heilemann, M. and Sauer, M. Nat Protoc More
Live-cell super-resolution imaging with trimethoprim conjugates 2010 Wombacher, R., M. Heidbreder, S. van de Linde, M.P. Sheetz, M. Heilemann, Cornish, V. W. and Sauer, M. Nat Methods More
The requirements for sub-diffraction resolution in continuous samples 2009 Heintzmann, R. and Gustafsson, M.G.L. Nat Photonics More
Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes 2008 Heilemann, M., van de Linde, S., Schüttpelz, M., Kasper, R., Seefeldt, B., Mukherjee, A., Tinnefeld, P. and Sauer, M. Angew Chem Int Ed More
Superresolution by localization of quantum dots using blinking statistics 2005 Lidke, K.A., Rieger, B., Jovin, T.M. and Heintzmann, R. Opics Express More