Live-Cell STED Microscopy with Genetically Encoded Biosensor.

Of the various super-resolution techniques, stimulated emission depletion (STED) microscopy achieves the best temporal resolution at high spatial resolution, enabling live-cell imaging beyond the diffraction limit. However, STED and most other super-resolution imaging methods utilize a particular type of information extractable from the raw data, namely the positions of fluorophores. To expand on the use of super-resolution techniques, we report here the live-cell STED microscopy of a dynamic biosensor. Using the fluorescent H2O2 sensor HyPer2 for subdiffraction imaging, we were able not only to image filaments with superior resolution by localizing emission but also to trace H2O2 produced within living cell by monitoring brightness of the probe. STED microscopy of HyPer2 demonstrates potential utility of FP-based biosensors for super-resolution experiments in situ and in vivo.

[1]  Carsten Schultz,et al.  Does cellular hydrogen peroxide diffuse or act locally? , 2011, Antioxidants & redox signaling.

[2]  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.

[3]  S. Lukyanov,et al.  HyPer-3: a genetically encoded H(2)O(2) probe with improved performance for ratiometric and fluorescence lifetime imaging. , 2013, ACS chemical biology.

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

[5]  Stefan W. Hell,et al.  Single-molecule STED microscopy with photostable organic fluorophores. , 2010, Small.

[6]  S. Rhee,et al.  H2O2, a Necessary Evil for Cell Signaling , 2006, Science.

[7]  Robert H Newman,et al.  Genetically encodable fluorescent biosensors for tracking signaling dynamics in living cells. , 2011, Chemical reviews.

[8]  S. Lukyanov,et al.  Genetically encoded fluorescent indicator for intracellular hydrogen peroxide , 2006, Nature Methods.

[9]  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 .

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

[11]  C. Winterbourn,et al.  Reconciling the chemistry and biology of reactive oxygen species. , 2008, Nature chemical biology.

[12]  C. Winterbourn,et al.  The High Reactivity of Peroxiredoxin 2 with H2O2 Is Not Reflected in Its Reaction with Other Oxidants and Thiol Reagents* , 2007, Journal of Biological Chemistry.

[13]  H. Sikes,et al.  Quantifying intracellular hydrogen peroxide perturbations in terms of concentration , 2014, Redox biology.

[14]  S. Rhee,et al.  Reversible Inactivation of Protein-tyrosine Phosphatase 1B in A431 Cells Stimulated with Epidermal Growth Factor* , 1998, The Journal of Biological Chemistry.

[15]  L. M. Vinokurov,et al.  A genetically encoded sensor for H2O2 with expanded dynamic range. , 2011, Bioorganic & medicinal chemistry.

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

[17]  J. Denu,et al.  Specific and reversible inactivation of protein tyrosine phosphatases by hydrogen peroxide: evidence for a sulfenic acid intermediate and implications for redox regulation. , 1998, Biochemistry.

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

[19]  Prabuddha Sengupta,et al.  Visualizing cell structure and function with point-localization superresolution imaging. , 2012, Developmental cell.

[20]  Toshiyuki Fukada,et al.  Reversible oxidation and inactivation of protein tyrosine phosphatases in vivo. , 2002, Molecular cell.

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

[22]  O. Lorenzo,et al.  Differential redox regulation within the PTP superfamily. , 2007, Cellular signalling.

[23]  Z. A. Wood,et al.  Structure, mechanism and regulation of peroxiredoxins. , 2003, Trends in biochemical sciences.

[24]  J. Stone.  An assessment of proposed mechanisms for sensing hydrogen peroxide in mammalian systems. , 2004, Archives of biochemistry and biophysics.

[25]  Christian Eggeling,et al.  STED microscopy of living cells – new frontiers in membrane and neurobiology , 2013, Journal of neurochemistry.

[26]  G Ulrich Nienhaus,et al.  Fluorescent proteins for live-cell imaging with super-resolution. , 2014, Chemical Society reviews.