WIMM PI Curriculum Vitae Personal Data Name Nationality Email Christian Eggeling Germany christian.eggeling@rdm.ox.ac.uk Present Position 10.2012-present Professor for Molecular Immunology / Group Leader MRC Human Immunology Unit (HIU) and Scientific Director Wolfson Imaging Centre Oxford, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom Previous Positions 1996 PhD theses Georg-August University Göttingen, Germany; Topic: “Analysis of photochemical kinetic and molecular dynamic through multi-dimensional singlemolecule fluorescence spectroscopy”, realized at the Max-Planck-Institute for Biophysical Chemistry Göttingen, Germany, group Dr. C. Seidel, director Prof. J. Troe, graded summa cum laude, awarded Otto-Hahn medal of the Max-Planck Society. 1996 Researcher in the group of Prof. R. Rigler at the Karolinska-Institute in Stockholm, Sweden (Fluorescence-Correlation-Spectroscopy). 2000 Scientist at the company Evotec OAI AG Hamburg, Germany: Development of novel (single-molecule based) fluorescence microscopy/spectroscopy techniques for highthroughput drug screening. 2004 Scientist at the department NanoBiophotonics, Max-Planck-Institute for Biophysical Chemistry Göttingen, Germany, department Prof. Hell: Development of novel highresolution fluorescence microscopy/spectroscopy techniques; 6.2011 awarded Nernst-Haber-Bodenstein-Prize of the German Bunsen Society. Research Achievements The determination of details of complex biological processes requires precise, sensitive and non-invasive detection methods. A lot of experiments apply fluorescence as a read-out parameter. Here, only parts of the sample are specifically stained with a fluorescent tag such as organic dyes or fluorescent proteins, and their fluorescence emission detected. The informational content of the fluorescence light is tremendous, stemming from its precise temporal and spatial registration along with the plenitude of read-out parameters (intensity, colour, etc.) and its sensitivity on environmental changes. Techniques such as (confocal) far-field microscopy allow a non-invasive observation of molecular distributions and characteristics of living samples, and of their dynamics in three-dimensional space. However, due to the diffraction of light the spatial resolution of far-field microscopy reaches only spatial scales down to about half the wavelength of light, i.e., ~200 nm, which is still far away from the molecular building blocks such as the size of a protein. Further, the signal strength, temporal resolution and observation time of common fluorescent labels is limited by their physical and chemical characteristics. These limitations often impede the precise disclosure of, for example, disease-relevant processes. My scientific activities so far aimed at the disclosure of characteristics influencing the registration of fluorescence signal and consequently the optimization of the sensitivity of fluorescence microscopy and spectroscopy experiments, and their use in biophysical applications, ranging from pioneer developments in the detection of single molecules, novel insights into the photophysics and –chemistry of fluorescent labels and the outline of optimization pathways, industrial application of (singlemolecule based) fluorescence microscopy such as its use in high-throughput screening for drug discovery, and most importantly the development of far-field fluorescence nanoscopy, i.e. the far-field microscopy with unlimited spatial resolution and its use to reveal novel insights into (live-cell) membrane biophysics and immunological processes. What are the Future Aims of Your Current Group? The main research interests of my laboratory will be focused on the application and development of ultra-sensitive, live-cell fluorescence microscopy techniques with a spatial resolution down to the molecular level (super-resolution microscopy or nanoscopy), superior to conventional optical microscopes. These super-resolution microscopes deliver a spatial resolution of down to below 40 nm in the living cell and, as a consequence, details of cellular structures and protein aggregations can be imaged and analysed with much larger details. I will further optimize these microscopes for their use to unravel nanoscopic changes at the molecular level in living cells following cellular immune responses. I will be planning to visualize previously un-detectable molecular interactions (such as protein-protein and protein-lipid interactions), which will shed new light on different molecular pathways triggered at the cell surface and intra-cellular during antigen presentation by dendritic cells and T-cell activation. For example, many cellular responses lead to subtle changes on the molecular level, demanding not only for a superior spatial resolution of the analysing method but also for the sensitivity to monitor single molecules over time and space. The combination of STED microscopy with single-molecule sensitive fluorescence-detection tools such as Fluorescence Correlation Spectroscopy (FCS) as well as the fast spatio-temporal tracking of single labelled molecules (single-particle tracking, SPT) allows for the disclosure of complex dynamical processes otherwise impeded by the limited spatial resolution of conventional farfield microscopy. STED-FCS or SPT offered us to gain novel insights into important cellular processes, such as lipid-lipid, lipid-protein, and protein-protein interactions and the formation of so-called “lipid-rafts” in the cellular plasma membrane. These molecular interactions play an important role in the cellular immune response. I will therefore apply and further develop the STED-FCS and SPT nanoscopy techniques to highlight important molecular processes on the plasma membrane as well as inside the cell during immunological reactions. How do These Aims Contribute to the Understanding and/or Management of Human Disease? The ultimate aim of my research is to address important questions in human health. So many important details in disease-related cellular processes could so far not accurately be observed due to limits in fluorescence microscopy such as its limited spatial resolution. Over the past years we have shown that one can overcome this limitation by using fluorescence nanoscopes and that these techniques really work for observing the living cells. However, so far the strengths of these novel tools for getting new insights in important biological and medical quests has not been revealed. By setting up these novel microscopes in the WIMM and collaborating with several scientists of the WIMM (for example Simon Davis and Vincenzo Cherundolo (T-cell activation)), i.e. by combining technological and biomedical knowhow, the potential for realizing major steps in life science and biomedical research is large: the observation of subtle, nanoscopic molecular processes in the living cell due to immunological reactions, viral attacks or cancer will reveal unprecedented new insights into the understanding of diseases and thus potentials for treating those. For example, an ultimate goal of my research would be to build up new technology platforms for drug discovery (if possible in a high throughput format), which all will have severe impact in medical research. Lay Summary of Research The understanding of cellular processes, for example, due to responses of the immune system requires the observation of structures down to the molecular level, such as how individual proteins arrange or interplay. For example, do certain proteins cluster following an immune response? An important issue is that these investigations have to be non-invasive, i.e. no response should be induced by the observation. Therefore, optical light microscopy is often used as a tool to image the arrangement of certain molecules/proteins in the cell, since light has a minimal influence on the studied system. Unfortunately, the spatial resolution of conventional light microscopes is limited, i.e. objects closer together than approximately 200 nm cannot be distinguished, small details of molecular organization appear blurred in an image, and it can, for example, not accurately be determined whether proteins cluster. In the recent years, we have developed microscopes that overcome this limitation and allow observing the living cell with so far unprecedented resolution. Consequently, applying these tools to biomedical studies such as the investigation of subtle changes during cellular immunological responses will realize new understandings of disease-related processes and pave new ways of drug development. All Publications Over the Past 5 Years *: corresponding author Peer reviewed articles C. Eggeling*, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova. V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, S. W. Hell Direct observation of the nanoscale dynamics of membrane lipids in a living cell. Nature 457, 1159-1163 (2009). E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, S. W. Hell Stimulated emission depletion microscopy reveals crystal colour centres with nanometric resolution. Nature Photonics 3, 144-147 (2009). G. Donnert, C. Eggeling*, S. W. Hell Triplet-relaxation microscopy with bunched pulsed excitation, Photochem. Photobiol. Sci. 8, 481-485 (2009). K.Y. Han, K. I. Willig, E. Rittweger, F. Jelezko, C. Eggeling*, S. W. Hell Three-Dimensional Stimulated Emission Depletion Microscopy of Nitrogen-Vacancy Centers in Diamond Using Continuous-Wave Light, Nano Lett. 9, 3323 – 3329 (2009). S.M. Polyakova, V.N. Belov, S.F. Yan, C. Eggeling, C. Ringemann, G. Schwarzmann, A. de Meijere, S.W. Hell New GM1 Ganglioside Derivatives for Selective Single and Double Labelling, Eur. JOC 30, 5162 – 5177 (2009). C. Ringemann, B. Harke, C. von Middendorff, R. Medda, A. Honigmann, R. Wagner, M. Leutenegger, A. Schönle, S.W. Hell, C. Eggeling* Exploring single-molecule dynamics with fluorescence nanoscopy, New J. Physics 11, 103054 (2009). K. Kolmakov, V.N. Belov, J. Bierwagen, C. Ringemann, V. Müller, C. Eggeling, S.W. Hell Red-Emitting Rhodamine Dyes for Fluorescence Microscopy and Nanoscopy, Chemistry – A Eur. J. 16, 158 – 166 (2010). G. Y. Mitronova, V.N. Belov, M.L. Bossi, C.A. Wurm, L. Meyer, R. Medda, G. Moneron, S. Bretschneider, C. Eggeling*, S. Jakobs, S.W. Hell New Fluorinated Rhodamines for Optical Microscopy and Nanoscopy, Chem Eur. J. 16, 4477 – 4488 (2010). T. Brakemann, G. Weber, M. Andresen, G. Groenhof, A.C. Stiel, S. Trowitzsch, C. Eggeling, H. Grubmüller, S.W. Hell, M.C. Wahl, S. Jakobs Molecular basis of the lightdriven switching of the photochromic fluorescent protein Padron, J. Biol. Chem. 285, 14603 – 14609 (2010). S. J. Sahl, M. Leutenegger, M. Hilbert, S. W. Hell, C. Eggeling* Fast molecular tracking maps nanoscale dynamics of plasma membrane lipids, Proc. Natl. Acad. Soc. USA 107, 6829 – 6834 (2010). K.Y. Han, S.K. Kim, C. Eggeling*, S. W. Hell Metastable Dark States Enable Ground State Depletion Microscopy of Nitrogen Vacancy Centers in Diamond with DiffractionUnlimited Resolution, Nano Lett. 10, 3199 – 3203 (2010). K. Kolmakov, V.N. Belov, C. A. Wurm, B. Harke, M. Leutenegger, C. Eggeling, S.W. Hell A Versatile Route to Red-Emitting Carbopyronine Dyes for Optical Microscopy and Nanoscopy, Eur. J. Org. Chem. 2010, 3593 – 3610 (2010). A. Honigmann, C. Walter, F. Erdmann, C. Eggeling, R. Wagner Characterization of Horizontal Lipid Bilayers as a Model System to Study Lipid Phase Separation, Biophys. J. 98, 2886 – 2894 (2010). I. Testa, C.A. Wurm, R. Medda, E. Rothermel, C. von Middendorf , J. Fölling, S. Jakobs, A. Schönle, S.W. Hell, C. Eggeling* Multicolor fluorescence nanoscopy in fixed and living cells by exciting conventional fluorophores with a single wavelength, Biophys. J. 99, 2686 - 2694 (2010). J. Bierwagen, I. Testa, J. Fölling, D. Wenzel, S. Jakobs, C. Eggeling, S.W. Hell Far-Field Autofluorescence Nanoscopy, Nano Lett. 10, 4249 – 4252 (2010). M. Leutenegger, C. Eggeling, S.W. Hell (2010) Analytical description of STED microscopy performance, Opt. Expr. 18, 26417 – 26429 (2010). G. Vicidomini, G. Moneron, K.Y. Han, V. Westphal, H. Ta, M. Reuss, J. Engelhardt, C. Eggeling, S.W. Hell Sharper low-power STED nanoscopy by time gating, Nature Meth. 8, 571 – 573 (2011). T. Brakemann, A.C. Stiel, G. Weber, M. Andresen, I. Testa, T. Grotjohann, M. Leutenegger, U. Plessmann, H. Urlaub, C. Eggeling, M. C. Wahl, S.W. Hell, S. Jakobs A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching, Nature Biotechnol. 29, 942 – 947 (2011). T. Grotjohann, I. Testa, M. Leutenegger, H. Bock, N. T. Urban, F. Lavoie-Cardinal, K.I. Willig, C. Eggeling, S. Jakobs, S.W. Hell Diffraction-unlimited all-optical imaging and writing with a photochromic GFP, Nature 478, 204 – 208 (2011). V. Mueller, C. Ringemann, A. Honigmann, G. Schwarzmann, R. Medda, M. Leutenegger, S. Polyakova, V.N. Belov, S.W. Hell, C. Eggeling* STED Nanoscopy Reveals Molecular Details of Cholesterol- and Cytoskeleton-Modulated Lipid Interactions in Living Cells, Biophys. J. 101, 1651 – 1660 (2011). G. Vicidomini, G. Moneron, C. Eggeling, E. Rittweger, S.W. Hell, STED with wavelengths closer to the emission maximum, Opt. Expr. 20, 5225 – 5236 (2012). M. Leutenegger, C. Ringemann, T. Lasser, S.W. Hell, C. Eggeling* Fluorescence correlation spectroscopy with a total internal reflection fluorescence STED microscope (TIRF-STED-FCS), Opt. Expr. 20, 5243 – 5263 (2012). E. Szegin, I. Levental, M. Grzybek, G. Schwarzmann, V. Mueller, A. Honigmann, V.N. Belov, C. Eggeling, Ü. Coskun, K. Simons, P. Schwille Partitioning, diffusion, and ligand binding of raft lipid analogs in model and cellular plasma membranes, BBA Biomembranes 1818, 1777–1784 (2012). I. Testa, N. T. Urban, S. Jakobs, C. Eggeling, K. I. Willig, S. W. Hell Nanoscopy of Living Brain Slices with Low Light Levels, Neuron 75, 992 – 1000 (2012) M. Bally, G. E. Rydell, R. Zahn, W. Nasir, C. Eggeling, M. E. Breimer, L. Svensson, F. Hook, G. Larson Norovirus GII.4 Virus-like Particles Recognize Galactosylceramides in Domains of Planar Supported Lipid Bilayers, Angewandte Chemie Int Edition 51, 12020 –12024 (2012) K. Y. Han, D. Wildanger, E. Rittweger, J. Meijer, S. Pezzagna, S. W. Hell, C. Eggeling* Dark state photophysics of diamond nitrogen-vacancy centres, New J Physics 14, 123002 (2012) T. Grotjohann, I. Testa, M. Reuss, T. Brakemann, C. Eggeling, S. W. Hell, S. Jakobs rsEGFP2 enables fast RESOLFT nanoscopy of living cells, eLife 1:e00248 (2012) A. Honigmann, V. Mueller, S. W. Hell, C. Eggeling* STED microscopy detects and quantifies liquid phase separation in lipid membranes using a new far-red emitting fluorescent phosphoglycerolipid analogue, Faraday Discussion 161, 77–89 (2013) G. Lukinavičius, K. Umezawa, N. Olivier, A. Honigmann, G. Yang, T. Plas, V. Mueller, L. Reymond, I. R. Corrêa Jr, Z.-G. Luo, C. Schultz, E. A. Lemke, P. Heppenstall, C. Eggeling, S. Manley, K. Johnsson A near-infrared fluorophore for live-cell superresolution microscopy of cellular proteins, Nature Chemistry 5, 132-139 (2013) G. Vicidomini, A. Schönle, H. Ta, K. Y. Han, G. Moneron, C. Eggeling, S. W. Hell STED Nanoscopy with Time-Gated Detection: Theoretical and Experimental Aspects, PLOS one 8, e54421 (2013) A. Honigmann, G. van den Bogaart, E. Iraheta, H. J. Risselada, D. Milovanovic, V. Mueller, S. Müllar, U. Diederichsen, D. Fasshauer, H. Grubmüller, S. W. Hell, C. Eggeling, K. Kühnel, R. Jahn, Phosphatidylinositol 4,5-bisphosphate clusters act as molecular beacons for vesicle recruitment, Nature Structural Molecular Biology 20, 679-686 (2013) A. Chmyrov, J. Keller, T. Grotjohann, M. Ratz, E. d'Este, S. Jakobs, C. Eggeling, S. W. Hell, Nanoscopy with more than 100,000 ‘doughnuts’, Nat Methods 10, 737–740 (2013) M. Solanko, A. Honigmann, H. S. Midtiby, F. W. Lund, J. Brewer, V. Dekaris, R. Bittman, C. Eggeling, D. Wüstner, Membrane orientation and lateral diffusion of BODIPY-cholesterol as a function of probe structure, Biophys J in press (2013) M. P. Clausen, S. Galiani, J. Bernardino de la Serna, M. Fritzsche, J. Chojnacki, K. Gehmlich, B. C. Lagerholm, C. Eggeling* Pathways to optical STED microscopy, NanoBioImaging 1, 1-12 (2013) S. J. Sahl, M. Leutenegger, S. W. Hell, C. Eggeling* High-Resolution Tracking of SingleMolecule Diffusion in Membranes by Confocalized and Spatially Differentiated Fluorescence Photon Stream Recording, ChemPhysChem 15, 771-783 (2014) C. Guzmán, M. Šolman, A. Ligabue, O. Blaževitš, D. M. Andrade, L. Reymond, C. Eggeling, D. Abankwa The efficacy of Raf kinase recruitment to the GTPase H-ras depends on Hras membrane conformer specific nanoclustering, J Biol Chem 289, 9519-9533 (2014) A. Honigmann, S. Sadeghi, J. Keller, S. W. Hell, C. Eggeling, R. Vink A lipid bound actin meshwork organizes liquid phase separation in model membranes, eLife e01671 (2014) A. Schoenle, C. V. Middendorff, C. Ringemann, S. W. Hell, C. Eggeling Monitoring triplet state dynamics with fluorescence correlation spectroscopy: bias and correction. Microscopy Research Techniques 77, 528-536 (2014) S. K. Saka, A. Honigmann, C. Eggeling, S. W. Hell, T. Lang, S. O. Rizzol Multi-protein assemblies underlie the mesoscale organization of the plasma membrane. Nature Communications 5, 4509 (2014) Review Article C. Eggeling Fluoreszenz-Spektroskopie auf Nanoskalen: Die Aufdeckung von zellulären Membran-Dynamiken mit Hilfe der optischen STED Mikroskopie und Spektroskopie. Bunsenmagazin 6, 205-213 (2011) C. Eggeling STED-FCS Nanoscopy of Membrane Dynamics. In: Fluorescent Methods to Study Biological Membranes; Eds.: Y. Mely, G. Duportail; Springer Series on Fluorescence Vol. 13; Springer-Verlag, Berlin, 291 – 309 (2012) V. Mueller, A. Honigmann, C. Ringemann, R. Medda, G. Schwarzmann, C. Eggeling FCS in STED Microscopy: Studying the Nanoscale of Lipid Membrane Dynamics. In: Methods in Enzymology, Vol. 519; Ed: S. Y. Tetin; Burlington: Academic Press, 1 – 38 (2013) C. Eggeling, K. I. Willig, F. J. Barrantes STED microscopy of living cells - New frontiers in membrane and neurobiology, J Neurochem 126, 203–212 (2013) C. Eggeling, A. Honigmann Molecular Plasma Membrane Dynamics Dissected by STED Nanoscopy and Fluorescence Correlation Spectroscopy (STED-FCS). In: Cell Membrane Nanodomains: from Biochemistry to Nanoscopy; Ed: D. S. Lidke, A. Cambi; CRC Press (2014) M. A. Lauterbach, C. Eggeling Foundations of Sted Microscopy. Neuromethods 86, 41-71 (2014) C. Eggeling, M. Heilemann Editorial overview: Molecular imaging. Current Opinion Chemical Biolology 20, v–vii (2014) Proceedings and Other Publications A. Honigmann, C. Eggeling, M. Schulze, A. Lepert Super-resolution STED microscopy advances with yellow CW OPSL. LaserFocusWorld 48, 75-79 (2012). V. Mueller, C. Eggeling, H. Karlsson, D. von Gegerfelt, CW DPSS Lasers Make STED Microscopy More Practical, Biophotonics 19, 30-32 (2012). C. Eggeling, A. Honigmann, M. Schulze, gSTED Microscopy with an OPSL: Cutting Edge Super-Resolution. Optik & Photonik 7, 44–46 (2012) A. Honigmann, V. Mueller, U. P. Fernando, C. Eggeling, J. Sperling Simplyfing STED Microscopy of Photostable Red-Emitting Labels. Laser + Potonik. 5, 40-42 (2013) Ten Key Publications Throughout your Career C. Eggeling, J. Widengren, R. Rigler, C. A. M. Seidel, Photobleaching of fluorescent dyes under conditions used for single-molecule detection: Evidence of two-step photolysis. Anal. Chem. 70, 2651-2659 (1998). C. Eggeling, L. Brand, D. Ullmann, S. Jäger, Highly sensitive fluorescence detection technology currently available for HTS. Drug Discovery Today 8, 632-641 (2003). M. Hofmann, C. Eggeling, S. Jakobs, S. W. Hell, Breaking the diffraction barrier in fluorescence microscopy at low light intensities using reversibly photoswitchable proteins. Proc. Natl. Acad. Sci. USA 102, 17565-17569 (2006). G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, S. W. Hell, Macromolecular-scale resolution in biological fluorescence microscopy. Proc. Natl. Acad. Soc. USA 103, 11440-11445 (2006). J. J. Sieber, K. I. Willig, C. Kutzner, C. Gerding-Reimers, B. Harke, G. Donnert, B. Rammner, C. Eggeling, S. W. Hell, H. Grubmüller, T. Lang, Anatomy and dynamics of a supramolecular membrane protein cluster. Science 317, 1072-1076 (2007). J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling*, S. W. Hell Fluorescence nanoscopy by ground-state depletion and single-molecule return. Nature Meth. 5, 943-945 (2008). C. Eggeling*, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova. V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, S. W. Hell Direct observation of the nanoscale dynamics of membrane lipids in a living cell. Nature 457, 1159-1163 (2009). S. J. Sahl, M. Leutenegger, M. Hilbert, S. W. Hell, C. Eggeling* Fast molecular tracking maps nanoscale dynamics of plasma membrane lipids, Proc. Natl. Acad. Soc. USA 107, 6829 – 6834 (2010). G. Vicidomini, G. Moneron, K.Y. Han, V. Westphal, H. Ta, M. Reuss, J. Engelhardt, C. Eggeling, S.W. Hell Sharper low-power STED nanoscopy by time gating, Nature Meth. 8, 571 – 573 (2011). T. Grotjohann, I. Testa, M. Leutenegger, H. Bock, N. T. Urban, F. Lavoie-Cardinal, K.I. Willig, C. Eggeling, S. Jakobs, S.W. Hell Diffraction-unlimited all-optical imaging and writing with a photochromic GFP, Nature 478, 204 – 208 (2011). Markers of Esteem 1999 Otto-Hahn medal of the Max-Planck society rewarding scientific achievements during the PhD work 2006 Cozarelli award of the Proceedings of the National Academy of Sciences USA for one of the best publications of the year 2006 2011 Nernst-Haber-Bodenstein Prize of the Bunsen Society (German Physical Chemistry Society) Current Grant Support Sub-project A6 (Pattern formation in membranes with quenched disorder) within the Collaborative Research Center (SFB) 937 of the University Göttingen (since 9.2010). Sub-project B11 (Nanoscale observations of membrane dynamics) with the Collaborative Research Center (SFB) 755 of the University Göttingen (since 2011). Next Generation Optical Microscopy – Nanoscopy Oxford, MRC/EPSRC/BBSRC (with Micron/Biochemistry Oxford) (since 3.2013) Dynamics of Peroxisomal Protein Transport, BBSRC (since 4.2013) Marie-Curie Career Integration Grant (contribution to group member Jorge Bernardino de la Serna) (since 10.2013) EMBO Long-Term Fellowship/Marie-Curie Fellowship (contribution to group member Erdinc Sezgin) (since 1.2014) Wellcome Trust Enhancement of Strategic Award (091911) entitled: Advanced Microscopy for Chromosome and RNA dynamics (with Micron/Biochemistry and Chemistry Oxford) (since 3.2014) Wellcome Trust Institutional Strategic Support Fund (WTISSF) 2013-2014 entitled: Containment level 3 super-resolution STED imaging of live virus host-cell interactions (with Lucy Dorrell/NDM Oxford) (since 6.2014) EP Abraham Cephalosporin Trust Fund 2014 entitled: Nanoscopy Oxford: Super-resolution optical STED microscope facility at the WIMM (since 6.2014) Wellcome Trust Multi-User Equipment Grant entitled: Advanced super-resolution fluorescence STED microscopy of the cellular interior (since 07.2014)