STED microscopy of living cells – new frontiers in membrane and neurobiology

Recent developments in fluorescence far‐field microscopy such as STED microscopy have accomplished observation of the living cell with a spatial resolution far below the diffraction limit. Here, we briefly review the current approaches to super‐resolution optical microscopy and present the implementation of STED microscopy for novel insights into live cell mechanisms, with a focus on neurobiology and plasma membrane dynamics.

[1]  Suliana Manley,et al.  Putting super-resolution fluorescence microscopy to work , 2008, Nature Methods.

[2]  S. Hell,et al.  STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis , 2006, Nature.

[3]  Christian Eggeling,et al.  Metastable dark States enable ground state depletion microscopy of nitrogen vacancy centers in diamond with diffraction-unlimited resolution. , 2010, Nano letters.

[4]  M. Wallace,et al.  Lucky imaging: improved localization accuracy for single molecule imaging. , 2009, Biophysical journal.

[5]  M. Heilemann,et al.  Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes. , 2008, Angewandte Chemie.

[6]  J. Rothman,et al.  Two-color STED microscopy in living cells , 2011, Biomedical optics express.

[7]  Christian Eggeling,et al.  Exploring single-molecule dynamics with fluorescence nanoscopy , 2009 .

[8]  D. Evanko.  Primer: fluorescence imaging under the diffraction limit , 2008, Nature Methods.

[9]  C. Eggeling.  STED-FCS Nanoscopy of Membrane Dynamics , 2012 .

[10]  Keith A. Lidke,et al.  Simultaneous multiple-emitter fitting for single molecule super-resolution imaging , 2011, Biomedical optics express.

[11]  S. Hell,et al.  Spherical nanosized focal spot unravels the interior of cells , 2008, Nature Methods.

[12]  S. Hell.  Microscopy and its focal switch , 2008, Nature Methods.

[13]  Ji Yu,et al.  Investigating Sub-Spine Actin Dynamics in Rat Hippocampal Neurons with Super-Resolution Optical Imaging , 2009, PloS one.

[14]  Gael Moneron,et al.  Two-photon excitation STED microscopy. , 2009, Optics express.

[15]  M. Dyba,et al.  In vivo labeling method using a genetic construct for nanoscale resolution microscopy. , 2009, Biophysical journal.

[16]  E. Betzig,et al.  Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics , 2008, Nature Methods.

[17]  S. Hell,et al.  Sharper low-power STED nanoscopy by time gating , 2011, Nature Methods.

[18]  Katharina Landfester,et al.  Characterization via two-color STED microscopy of nanostructured materials synthesized by colloid electrospinning. , 2011, Langmuir : the ACS journal of surfaces and colloids.

[19]  Christian Eggeling,et al.  A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching , 2011, Nature Biotechnology.

[20]  S. Holden,et al.  DAOSTORM: an algorithm for high- density super-resolution microscopy , 2011, Nature Methods.

[21]  J. Lippincott-Schwartz,et al.  Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure , 2009, Proceedings of the National Academy of Sciences.

[22]  Petra Schwille,et al.  Ultrasensitive investigations of biological systems by fluorescence correlation spectroscopy. , 2003, Methods.

[23]  S. Hell,et al.  Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. , 1994, Optics letters.

[24]  S. Hell,et al.  Nanoscale resolution in GFP-based microscopy , 2006, Nature Methods.

[25]  M. Gustafsson.  Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy , 2000, Journal of microscopy.

[26]  M. Gustafsson,et al.  Subdiffraction Multicolor Imaging of the Nuclear Periphery with 3D Structured Illumination Microscopy , 2008, Science.

[27]  Hervé Rigneault,et al.  Fluorescence correlation spectroscopy diffusion laws to probe the submicron cell membrane organization. , 2005, Biophysical journal.

[28]  Gerd Ulrich Nienhaus,et al.  Online image analysis software for photoactivation localization microscopy , 2009, Nature Methods.

[29]  Akihiro Kusumi,et al.  Paradigm shift of the plasma membrane concept from the two-dimensional continuum fluid to the partitioned fluid: high-speed single-molecule tracking of membrane molecules. , 2005, Annual review of biophysics and biomolecular structure.

[30]  Christian Eggeling,et al.  Fast molecular tracking maps nanoscale dynamics of plasma membrane lipids , 2010, Proceedings of the National Academy of Sciences.

[31]  Mark Bates,et al.  Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging , 2011, Nature Methods.

[32]  Michael A Thompson,et al.  Super-resolution imaging in live Caulobacter crescentus cells using photoswitchable EYFP , 2008, Nature Methods.

[33]  S. Hell,et al.  Ground-state-depletion fluorscence microscopy: A concept for breaking the diffraction resolution limit , 1995 .

[34]  S W Hell,et al.  STED nanoscopy reveals molecular details of cholesterol- and cytoskeleton-modulated lipid interactions in living cells. , 2011, Biophysical journal.

[35]  S. Hell,et al.  Imaging and writing at the nanoscale with focused visible light through saturable optical transitions , 2003 .

[36]  H. Flyvbjerg,et al.  Optimized localization-analysis for single-molecule tracking and super-resolution microscopy , 2010, Nature Methods.

[37]  L. Mets,et al.  Nanometer-localized multiple single-molecule fluorescence microscopy. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[38]  S. Hell,et al.  Focal spots of size lambda/23 open up far-field fluorescence microscopy at 33 nm axial resolution. , 2002, Physical review letters.

[39]  Andreas Volkmer,et al.  Molecular photobleaching kinetics of Rhodamine 6G by one- and two-photon induced confocal fluorescence microscopy. , 2005, Chemphyschem : a European journal of chemical physics and physical chemistry.

[40]  S. Weiss,et al.  Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI) , 2009, Proceedings of the National Academy of Sciences.

[41]  S. Hell,et al.  STED microscopy with continuous wave beams , 2007, Nature Methods.

[42]  S Wolter,et al.  Real‐time computation of subdiffraction‐resolution fluorescence images , 2010, Journal of microscopy.

[43]  R. Dobarzić,et al.  [Fluorescence microscopy]. , 1975, Plucne bolesti i tuberkuloza.

[44]  Beiträge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung , 1873 .

[45]  S. Hell,et al.  STED microscopy detects and quantifies liquid phase separation in lipid membranes using a new far-red emitting fluorescent phosphoglycerolipid analogue. , 2013, Faraday discussions.

[46]  Alexander R Small,et al.  Theoretical limits on errors and acquisition rates in localizing switchable fluorophores. , 2008, Biophysical journal.

[47]  R. Heintzmann,et al.  Superresolution by localization of quantum dots using blinking statistics. , 2005, Optics express.

[48]  S. Hell.  Toward fluorescence nanoscopy , 2003, Nature Biotechnology.

[49]  Thorsten Lang,et al.  Anatomy and Dynamics of a Supramolecular Membrane Protein Cluster , 2007, Science.

[50]  Christian Eggeling,et al.  Triplet-relaxation microscopy with bunched pulsed excitation , 2009, Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology.

[51]  Benjamin Harke,et al.  Three-dimensional nanoscopy of colloidal crystals. , 2008, Nano letters.

[52]  S. Hell,et al.  Two-color far-field fluorescence nanoscopy. , 2007, Biophysical journal.

[53]  W. Denk,et al.  Two-photon excitation in functional biological imaging. , 1996, Journal of biomedical optics.

[54]  Daniel R. Larson.  The economy of photons , 2010, Nature Methods.

[55]  S. Hell,et al.  Wide‐field subdiffraction RESOLFT microscopy using fluorescent protein photoswitching , 2007, Microscopy research and technique.

[56]  K. Chi.  Super-resolution microscopy: breaking the limits , 2008, Nature Methods.

[57]  Suliana Manley,et al.  Superresolution imaging using single-molecule localization. , 2010, Annual review of physical chemistry.

[58]  Y. Mély,et al.  Fluorescent Methods to Study Biological Membranes , 2013 .

[59]  S. Hess,et al.  Three-dimensional sub–100 nm resolution fluorescence microscopy of thick samples , 2008, Nature Methods.

[60]  Gerhard J Schütz,et al.  (Un)confined diffusion of CD59 in the plasma membrane determined by high-resolution single molecule microscopy. , 2007, Biophysical journal.

[61]  Keith A. Lidke,et al.  Fast, single-molecule localization that achieves theoretically minimum uncertainty , 2010, Nature Methods.

[62]  Christian Eggeling,et al.  rsEGFP2 enables fast RESOLFT nanoscopy of living cells , 2012, eLife.

[63]  Stefan W. Hell,et al.  Strategy for far-field optical imaging and writing without diffraction limit , 2004 .

[64]  M. Bruchez,et al.  STED nanoscopy in living cells using Fluorogen Activating Proteins. , 2009, Bioconjugate chemistry.

[65]  M. Gustafsson,et al.  Subdiffraction resolution in continuous samples , 2009 .

[66]  S. Hell,et al.  Stimulated emission depletion nanoscopy of living cells using SNAP-tag fusion proteins. , 2010, Biophysical journal.

[67]  Suliana Manley,et al.  A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins. , 2013, Nature chemistry.

[68]  J. J. Macklin,et al.  Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution , 2011, Proceedings of the National Academy of Sciences.

[69]  Christian Eggeling,et al.  Breaking the diffraction barrier in fluorescence microscopy by optical shelving. , 2007, Physical review letters.

[70]  Michael J Rust,et al.  Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM) , 2006, Nature Methods.

[71]  B. Chromy,et al.  Efficient maximum likelihood estimator fitting of histograms , 2010, Nature Methods.

[72]  Samuel J. Lord,et al.  Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function , 2009, Proceedings of the National Academy of Sciences.

[73]  Christian Eggeling,et al.  Super-resolution STED microscopy advances with yellow CW OPSL , 2012 .

[74]  S. Hell,et al.  Fluorescence nanoscopy by ground-state depletion and single-molecule return , 2008, Nature Methods.

[75]  Mark Bates,et al.  Three-Dimensional Super-Resolution Imaging by Stochastic Optical Reconstruction Microscopy , 2008, Science.

[76]  Peter Dedecker,et al.  Spectroscopic rationale for efficient stimulated-emission depletion microscopy fluorophores. , 2010, Journal of the American Chemical Society.

[77]  A. Ting,et al.  Fluorescent probes for super-resolution imaging in living cells , 2008, Nature Reviews Molecular Cell Biology.

[78]  U Valentin Nägerl,et al.  Two-color STED microscopy of living synapses using a single laser-beam pair. , 2011, Biophysical journal.

[79]  X. Zhuang,et al.  Fast three-dimensional super-resolution imaging of live cells , 2011, Nature Methods.

[80]  S. Hell,et al.  Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[81]  S. E. Irvine,et al.  Fast Sted Microscopy with Continuous Wave Fiber Lasers References and Links , 2022 .

[82]  Christian Eggeling,et al.  Diffraction-unlimited all-optical imaging and writing with a photochromic GFP , 2011, Nature.

[83]  W E Moerner,et al.  Single-molecule mountains yield nanoscale cell images , 2006, Nature Methods.

[84]  Måns Ehrenberg,et al.  Rotational brownian motion and fluorescence intensify fluctuations , 1974 .

[85]  Gael Moneron,et al.  Nanoscopy in a living multicellular organism expressing GFP. , 2011, Biophysical journal.

[86]  Akihiro Kusumi,et al.  Hierarchical organization of the plasma membrane: Investigations by single‐molecule tracking vs. fluorescence correlation spectroscopy , 2010, FEBS letters.

[87]  J. Rice,et al.  Beyond the diffraction limit: far-field fluorescence imaging with ultrahigh resolution. , 2007, Molecular bioSystems.

[88]  W. Denk,et al.  Two-photon laser scanning fluorescence microscopy. , 1990, Science.

[89]  Paul R Selvin,et al.  Polarization effect on position accuracy of fluorophore localization. , 2006, Optics express.

[90]  Steven Chu,et al.  Subnanometre single-molecule localization, registration and distance measurements , 2010, Nature.

[91]  J. Lippincott-Schwartz,et al.  High-density mapping of single-molecule trajectories with photoactivated localization microscopy , 2008, Nature Methods.

[92]  X. Zhuang,et al.  Breaking the Diffraction Barrier: Super-Resolution Imaging of Cells , 2010, Cell.

[93]  Bernardo L. Sabatini,et al.  Supraresolution Imaging in Brain Slices using Stimulated-Emission Depletion Two-Photon Laser Scanning Microscopy , 2009, Neuron.

[94]  Stefan W. Hell,et al.  Nanoscopy in a Living Mouse Brain , 2012, Science.

[95]  Joerg Bewersdorf,et al.  Far-red fluorescent protein excitable with red lasers for flow cytometry and superresolution STED nanoscopy. , 2010, Biophysical journal.

[96]  Stefan Bretschneider,et al.  Ground State Depletion Fluorescence Microscopy , 2008 .

[97]  Mike Heilemann,et al.  Subdiffraction-resolution fluorescence microscopy of myosin-actin motility. , 2010, Chemphyschem : a European journal of chemical physics and physical chemistry.

[98]  David A. Agard,et al.  Sevenfold improvement of axial resolution in 3D wide-field microscopy using two objective lenses , 1995, Electronic Imaging.

[99]  Marcus Dyba,et al.  Concepts for nanoscale resolution in fluorescence microscopy , 2004, Current Opinion in Neurobiology.

[100]  S. Hell,et al.  STED microscopy with a supercontinuum laser source. , 2008, Optics express.

[101]  M. Heilemann,et al.  Photoswitches: Key molecules for subdiffraction‐resolution fluorescence imaging and molecular quantification , 2009 .

[102]  A. Egner,et al.  Two-color far-field fluorescence nanoscopy based on photoswitchable emitters , 2007 .

[103]  P. Dedecker,et al.  Diffraction-unlimited optical microscopy , 2008 .

[104]  Christian Eggeling,et al.  Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[105]  S. Hell.  Far-Field Optical Nanoscopy , 2007, Science.

[106]  Christophe Zimmer,et al.  Super-Resolution Dynamic Imaging of Dendritic Spines Using a Low-Affinity Photoconvertible Actin Probe , 2011, PloS one.

[107]  S. Hell,et al.  Stimulated emission depletion (STED) nanoscopy of a fluorescent protein-labeled organelle inside a living cell , 2008, Proceedings of the National Academy of Sciences.

[108]  Thorsten Staudt,et al.  Sted Nanoscopy with Mass-produced Laser Diodes References and Links , 2022 .

[109]  Martin J Booth,et al.  Adaptive optics enables 3D STED microscopy in aberrating specimens. , 2012, Optics express.

[110]  M. Gustafsson.  Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[111]  J. Lippincott-Schwartz,et al.  Imaging Intracellular Fluorescent Proteins at Nanometer Resolution , 2006, Science.

[112]  W. Webb,et al.  Thermodynamic Fluctuations in a Reacting System-Measurement by Fluorescence Correlation Spectroscopy , 1972 .

[113]  Mark Bates,et al.  Super-resolution microscopy by nanoscale localization of photo-switchable fluorescent probes. , 2008, Current opinion in chemical biology.

[114]  R. Yuste,et al.  Morphological changes in dendritic spines associated with long-term synaptic plasticity. , 2001, Annual review of neuroscience.

[115]  Stefan W. Hell,et al.  Supporting Online Material Materials and Methods Figs. S1 to S9 Tables S1 and S2 References Video-rate Far-field Optical Nanoscopy Dissects Synaptic Vesicle Movement , 2022 .

[116]  T. Ha,et al.  Single-molecule high-resolution imaging with photobleaching. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[117]  Christian Eggeling,et al.  Nanoscopy of Living Brain Slices with Low Light Levels , 2012, Neuron.

[118]  L. Nguyen,et al.  Molecular layers underlying cytoskeletal remodelling during cortical development , 2010, Trends in Neurosciences.

[119]  R. Heintzmann,et al.  Saturated patterned excitation microscopy--a concept for optical resolution improvement. , 2002, Journal of the Optical Society of America. A, Optics, image science, and vision.

[120]  T. Bonhoeffer,et al.  Live-cell imaging of dendritic spines by STED microscopy , 2008, Proceedings of the National Academy of Sciences.

[121]  H. Brismar,et al.  Spatial distribution of Na+-K+-ATPase in dendritic spines dissected by nanoscale superresolution STED microscopy , 2011, BMC Neuroscience.

[122]  Rainer Heintzmann,et al.  Breaking the resolution limit in light microscopy. , 2013, Methods in cell biology.

[123]  Bo Huang,et al.  Super-resolution optical microscopy: multiple choices. , 2010, Current opinion in chemical biology.

[124]  Thorsten Staudt,et al.  Molecular orientation affects localization accuracy in superresolution far-field fluorescence microscopy. , 2011, Nano letters.

[125]  U Valentin Nägerl,et al.  STED nanoscopy of actin dynamics in synapses deep inside living brain slices. , 2011, Biophysical journal.

[126]  Dylan T Burnette,et al.  Bayesian localisation microscopy reveals nanoscale podosome dynamics , 2011, Nature Methods.

[127]  P. Schwille,et al.  Partitioning, diffusion, and ligand binding of raft lipid analogs in model and cellular plasma membranes. , 2012, Biochimica et biophysica acta.

[128]  A. Diaspro,et al.  Fast scanning STED and two‐photon fluorescence excitation microscopy with continuous wave beam , 2012, Journal of microscopy.

[129]  K. Chou,et al.  Subdiffraction-limit two-photon fluorescence microscopy for GFP-tagged cell imaging. , 2009, Biophysical journal.

[130]  S. Hell,et al.  Endosomal sorting of readily releasable synaptic vesicles , 2010, Proceedings of the National Academy of Sciences.

[131]  X. Zhuang,et al.  Superresolution Imaging of Chemical Synapses in the Brain , 2010, Neuron.

[132]  Mark Bates,et al.  Super-resolution fluorescence microscopy. , 2009, Annual review of biochemistry.

[133]  E. Abbe.  Beiträge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung , 1873 .

[134]  S. Hell,et al.  Direct observation of the nanoscale dynamics of membrane lipids in a living cell , 2009, Nature.

[135]  A. Kraegeloh,et al.  STED microscopy and its applications: new insights into cellular processes on the nanoscale. , 2012, Chemphyschem : a European journal of chemical physics and physical chemistry.

[136]  Hazen P. Babcock,et al.  Dual-objective STORM reveals three-dimensional filament organization in the actin cytoskeleton , 2011, Nature Methods.

[137]  Volker Westphal,et al.  A STED microscope aligned by design. , 2009, Optics express.

[138]  C. Seidel,et al.  Photobleaching of Fluorescent Dyes under Conditions Used for Single-Molecule Detection:  Evidence of Two-Step Photolysis. , 1998, Analytical chemistry.

[139]  Mark Bates,et al.  Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes , 2007, Science.

[140]  C. Zimmer,et al.  QuickPALM: 3D real-time photoactivation nanoscopy image processing in ImageJ , 2010, Nature Methods.

[141]  Kai Simons,et al.  Lipid Rafts As a Membrane-Organizing Principle , 2010, Science.

[142]  Michael D. Mason,et al.  Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. , 2006, Biophysical journal.