Super-Resolution Imaging Conditions for enhanced Yellow Fluorescent Protein (eYFP) Demonstrated on DNA Origami Nanorulers

Photostability is one of the crucial properties of a fluorophore which strongly influences the quality of single molecule-based super-resolution imaging. Enhanced yellow fluorescent protein (eYFP) is one of the most widely used versions of fluorescent proteins in modern cell biology exhibiting fast intrinsic blinking and reversible photoactivation by UV light. Here, we developed an assay for studying photostabilization of single eYFP molecules with respect to fast blinking and demonstrated a 6-fold enhanced photostability of single eYFP molecules with a beneficial influence on the blinking kinetics under oxygen removal and addition of aliphatic thiols (dSTORM-buffer). Conjugation to single stranded DNA and immobilization via DNA hybridization on a DNA origami 12 helix bundle in aqueous solution allowed photophyiscal studies of eYFP at the single-molecule level and at close to physiological conditions. The benefit of improved photophysical properties for localization-based super-resolution microscopy is demonstrated and quantitatively characterized by imaging 12 helix bundle DNA origami nanorulers with binding sites at designed distances of 160 and 100 nm and by imaging microtubules in fixed mammalian Vero cells.

[1]  Jeffrey R Moffitt,et al.  Characterization and development of photoactivatable fluorescent proteins for single-molecule–based superresolution imaging , 2014, Proceedings of the National Academy of Sciences.

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

[3]  P. Dedecker,et al.  Fluorescent proteins: shine on, you crazy diamond. , 2013, Journal of the American Chemical Society.

[4]  Roger Y. Tsien,et al.  Crystal Structure of the Aequorea victoria Green Fluorescent Protein , 1996, Science.

[5]  Matthew D. Lew,et al.  Three-dimensional superresolution colocalization of intracellular protein superstructures and the cell surface in live Caulobacter crescentus , 2011, Proceedings of the National Academy of Sciences.

[6]  W. E. Moerner,et al.  The Fluorescence Dynamics of Single Molecules of Green Fluorescent Protein , 1999 .

[7]  Helge Ewers,et al.  Single-molecule localization microscopy using mCherry. , 2014, Chemphyschem : a European journal of chemical physics and physical chemistry.

[8]  C. Niemeyer,et al.  Synthesis of fluorescent oligonucleotide--EYFP conjugate: towards supramolecular construction of semisynthetic biomolecular antennae. , 2004, Organic & biomolecular chemistry.

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

[10]  Margaret Comerford Freda.  The solution? , 2005, MCN. The American journal of maternal child nursing.

[11]  P. Tinnefeld,et al.  DNA origami–based standards for quantitative fluorescence microscopy , 2014, Nature Protocols.

[12]  K. Lukyanov,et al.  Anti-Fading Media for Live Cell GFP Imaging , 2012, PloS one.

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

[14]  Taekjip Ha,et al.  Probing Cellular Protein Complexes via Single Molecule Pull-down , 2011, Nature.

[15]  R. Tsien,et al.  On/off blinking and switching behaviour of single molecules of green fluorescent protein , 1997, Nature.

[16]  P. Tinnefeld,et al.  Eukaryotic and archaeal TBP and TFB/TF(II)B follow different promoter DNA bending pathways , 2014, Nucleic acids research.

[17]  David Neff,et al.  NTA directed protein nanopatterning on DNA Origami nanoconstructs. , 2009, Journal of the American Chemical Society.

[18]  Adam H. Marblestone,et al.  Rapid prototyping of 3D DNA-origami shapes with caDNAno , 2009, Nucleic acids research.

[19]  Taekjip Ha,et al.  Photophysics of fluorescent probes for single-molecule biophysics and super-resolution imaging. , 2012, Annual review of physical chemistry.

[20]  Philip Tinnefeld,et al.  Fluorescence microscopy with 6 nm resolution on DNA origami. , 2014, Chemphyschem : a European journal of chemical physics and physical chemistry.

[21]  Konstantin A Lukyanov,et al.  Cell culture medium affects GFP photostability: a solution , 2009, Nature Methods.

[22]  Philip Tinnefeld,et al.  Choosing dyes for cw-STED nanoscopy using self-assembled nanorulers , 2014, Physical chemistry chemical physics : PCCP.

[23]  P. Rothemund.  Folding DNA to create nanoscale shapes and patterns , 2006, Nature.

[24]  Takeharu Nagai,et al.  Shift anticipated in DNA microarray market , 2002, Nature Biotechnology.

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

[26]  M. Heilemann,et al.  Live-cell super-resolution imaging with synthetic fluorophores. , 2012, Annual review of physical chemistry.

[27]  R. Tsien,et al.  Partitioning of Lipid-Modified Monomeric GFPs into Membrane Microdomains of Live Cells , 2002, Science.

[28]  Philip Tinnefeld,et al.  Fluorescence and super-resolution standards based on DNA origami , 2012, Nature Methods.

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

[30]  Clive R. Bagshaw,et al.  Protonation, photobleaching, and photoactivation of yellow fluorescent protein (YFP 10C): a unifying mechanism. , 2005, Biochemistry.

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

[32]  Jan Vogelsang,et al.  Make them blink: probes for super-resolution microscopy. , 2010, Chemphyschem : a European journal of chemical physics and physical chemistry.

[33]  R. Tsien,et al.  The Fluorescent Toolbox for Assessing Protein Location and Function , 2006, Science.

[34]  K. Timmis,et al.  Broad host range vectors for expression of proteins with (Twin-) Strep-tag, His-tag and engineered, export optimized yellow fluorescent protein , 2013, Microbial Cell Factories.

[35]  Christian Blum,et al.  Room temperature spectrally resolved single-molecule spectroscopy reveals new spectral forms and photophysical versatility of aequorea green fluorescent protein variants. , 2004, Biophysical journal.

[36]  Clemens F Kaminski,et al.  Correcting chromatic offset in multicolor super-resolution localization microscopy. , 2013, Optics express.

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

[38]  Jan Vogelsang,et al.  Superresolution microscopy on the basis of engineered dark states. , 2008, Journal of the American Chemical Society.

[39]  Paul W. K. Rothemund,et al.  Rothemund, P.W.K.: Folding DNA to create nanoscale shapes and patterns. Nature 440, 297-302 , 2006 .

[40]  Philipp C Nickels,et al.  DNA origami nanopillars as standards for three-dimensional superresolution microscopy. , 2013, Nano letters.

[41]  Yuichiro Hori,et al.  [Crystal structure of the Aequorea victoria green fluorescent protein]. , 2007, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme.

[42]  Peter J. Verveer,et al.  Chemically Induced Photoswitching of Fluorescent Probes—A General Concept for Super-Resolution Microscopy , 2011, Molecules.

[43]  Guido Caldarelli,et al.  Web Search Queries Can Predict Stock Market Volumes , 2011, PloS one.

[44]  Philip Tinnefeld,et al.  DNA origami as biocompatible surface to match single-molecule and ensemble experiments , 2012, Nucleic acids research.