Three-dimensional superresolution colocalization of intracellular protein superstructures and the cell surface in live Caulobacter crescentus

Recently, single-molecule imaging and photocontrol have enabled superresolution optical microscopy of cellular structures beyond Abbe’s diffraction limit, extending the frontier of noninvasive imaging of structures within living cells. However, live-cell superresolution imaging has been challenged by the need to image three-dimensional (3D) structures relative to their biological context, such as the cellular membrane. We have developed a technique, termed superresolution by power-dependent active intermittency and points accumulation for imaging in nanoscale topography (SPRAIPAINT) that combines imaging of intracellular enhanced YFP (eYFP) fusions (SPRAI) with stochastic localization of the cell surface (PAINT) to image two different fluorophores sequentially with only one laser. Simple light-induced blinking of eYFP and collisional flux onto the cell surface by Nile red are used to achieve single-molecule localizations, without any antibody labeling, cell membrane permeabilization, or thiol-oxygen scavenger systems required. Here we demonstrate live-cell 3D superresolution imaging of Crescentin-eYFP, a cytoskeletal fluorescent protein fusion, colocalized with the surface of the bacterium Caulobacter crescentus using a double-helix point spread function microscope. Three-dimensional colocalization of intracellular protein structures and the cell surface with superresolution optical microscopy opens the door for the analysis of protein interactions in living cells with excellent precision (20–40 nm in 3D) over a large field of view (12 × 12 μm).

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

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

[3]  Glen L. Beane,et al.  Experimental characterization of 3D localization techniques for particle-tracking and super-resolution microscopy. , 2009, Optics express.

[4]  Christian Eggeling,et al.  Macromolecular-scale resolution in biological fluorescence microscopy. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[5]  Lucy Shapiro,et al.  Systems biology of Caulobacter. , 2007, Annual review of genetics.

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

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

[8]  Matthew D Lew,et al.  Corkscrew point spread function for far-field three-dimensional nanoscale localization of pointlike objects. , 2011, Optics letters.

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

[10]  L. Shapiro,et al.  A comprehensive set of plasmids for vanillate- and xylose-inducible gene expression in Caulobacter crescentus , 2007, Nucleic acids research.

[11]  R. Hochstrasser,et al.  Super-resolution microscopy of lipid bilayer phases. , 2011, Journal of the American Chemical Society.

[12]  K. Neuman,et al.  Measurement of the effective focal shift in an optical trap. , 2005, Optics letters.

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

[14]  W E Moerner,et al.  Azido push-pull fluorogens photoactivate to produce bright fluorescent labels. , 2010, The journal of physical chemistry. B.

[15]  N. Cheng.  Formula for the Viscosity of a Glycerol−Water Mixture , 2008 .

[16]  M. L. Le Gros,et al.  X-ray tomography generates 3-D reconstructions of the yeast, saccharomyces cerevisiae, at 60-nm resolution. , 2003, Molecular biology of the cell.

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

[18]  W. P. Ambrose,et al.  Detection and spectroscopy of single pentacene molecules in a p‐terphenyl crystal by means of fluorescence excitation , 1991 .

[19]  J. Shaevitz,et al.  Effect of aberration on height calibration in three-dimensional localization-based microscopy and particle tracking. , 2009, Applied optics.

[20]  S. Hell,et al.  Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index , 1993 .

[21]  Jianyong Tang,et al.  Near-isotropic 3D optical nanoscopy with photon-limited chromophores , 2010, Proceedings of the National Academy of Sciences.

[22]  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.

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

[24]  Ned S. Wingreen,et al.  Self-Organization of the Escherichia coli Chemotaxis Network Imaged with Super-Resolution Light Microscopy , 2009, PLoS biology.

[25]  W. Moerner,et al.  Experimental and theoretical investigations of environmentally sensitive single-molecule fluorophores , 2004, InternationalQuantum Electronics Conference, 2004. (IQEC)..

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

[27]  J. Poindexter.  BIOLOGICAL PROPERTIES AND CLASSIFICATION OF THE CAULOBACTER GROUP , 1964, Bacteriological reviews.

[28]  Zemer Gitai,et al.  Single molecules of the bacterial actin MreB undergo directed treadmilling motion in Caulobacter crescentus. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[29]  Michael T. Laub,et al.  Cell-cycle progression and the generation of asymmetry in Caulobacter crescentus , 2004, Nature Reviews Microbiology.

[30]  Suliana Manley,et al.  Photoactivatable mCherry for high-resolution two-color fluorescence microscopy , 2009, Nature Methods.

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

[32]  L. Shapiro,et al.  A spindle-like apparatus guides bacterial chromosome segregation , 2010, Nature Cell Biology.

[33]  Paul R. Selvin,et al.  Myosin V Walks Hand-Over-Hand: Single Fluorophore Imaging with 1.5-nm Localization , 2003, Science.

[34]  R. Piestun,et al.  Three dimensional tracking of fluorescent microparticles using a photon-limited double-helix response system. , 2008, Optics express.

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

[36]  W E Moerner,et al.  Fluorescence correlation spectroscopy reveals fast optical excitation-driven intramolecular dynamics of yellow fluorescent proteins. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[37]  Ned S. Wingreen,et al.  Self-Organization of the Escherichia Coli Chemotaxis Network Imaged with Super-Resolution Light Microscopy , 2010 .

[38]  S. Hell,et al.  Two-color nanoscopy of three-dimensional volumes by 4Pi detection of stochastically switched fluorophores , 2011, Nature Methods.

[39]  Kristin L. Hazelwood,et al.  A bright and photostable photoconvertible fluorescent protein for fusion tags , 2009, Nature Methods.

[40]  X. Zhuang,et al.  Whole cell 3D STORM reveals interactions between cellular structures with nanometer-scale resolution , 2008, Nature Methods.

[41]  J. Frydman,et al.  Probing the sequence of conformationally induced polarity changes in the molecular chaperonin GroEL with fluorescence spectroscopy. , 2005, The journal of physical chemistry. B.

[42]  Michael W. Davidson,et al.  Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes , 2007, Proceedings of the National Academy of Sciences.

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

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

[45]  C. Jacobs-Wagner,et al.  Bacterial intermediate filaments: in vivo assembly, organization, and dynamics of crescentin. , 2009, Genes & development.

[46]  R. Hochstrasser,et al.  Wide-field subdiffraction imaging by accumulated binding of diffusing probes , 2006, Proceedings of the National Academy of Sciences.

[47]  Rafael Piestun,et al.  Three-dimensional localization with nanometer accuracy using a detector-limited double-helix point spread function system , 2009 .

[48]  W. E. Moerner,et al.  Localizing and tracking single nanoscale emitters in three dimensions with high spatiotemporal resolution using a double-helix point spread function. , 2010, Nano letters.

[49]  W E Moerner,et al.  Nonlinear optical chromophores as nanoscale emitters for single-molecule spectroscopy. , 2005, Accounts of chemical research.

[50]  Matthew D. Lew,et al.  Three-dimensional localization precision of the double-helix point spread function versus astigmatism and biplane. , 2010, Applied physics letters.

[51]  Michael A Thompson,et al.  Super-resolution imaging of the nucleoid-associated protein HU in Caulobacter crescentus. , 2011, Biophysical journal.

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

[53]  C. Jacobs-Wagner,et al.  The Bacterial Cytoskeleton An Intermediate Filament-Like Function in Cell Shape , 2003, Cell.

[54]  V. Verkhusha,et al.  Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light , 2006, Nature Biotechnology.

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

[56]  Richard D. Leapman,et al.  Nanoscale 3D cellular imaging by axial scanning transmission electron tomography , 2009, Nature Methods.

[57]  W E Moerner,et al.  Diffusion of lipid-like single-molecule fluorophores in the cell membrane. , 2006, The journal of physical chemistry. B.

[58]  Samuel J. Lord,et al.  Modifications of DCDHF single molecule fluorophores to impart water solubility. , 2007, Tetrahedron letters.

[59]  Matthew D Lew,et al.  In vivo three-dimensional superresolution fluorescence tracking using a double-helix point spread function , 2010, BiOS.