Polarization sensitive, three-dimensional, single-molecule imaging of cells with a double-helix system.

Double-helix point spread function photoactivation-localization microscopy allows three-dimensional (3D) superresolution imaging of objects smaller than the optical diffraction-limit. We demonstrate polarization sensitive detection with 3D super-localization of single-molecules and unveil 3D polarization specific characteristics of single-molecules within the intracellular structure of PtK1 cells expressing photoactivatable green fluorescent protein. The system modulates orthogonal polarization components of single-molecule emissions with a single spatial light modulator and detects them separately with a single detector. Information obtained from the two polarization channels demonstrates polarization based contrast in 3D superresolution imaging. Further, we show that the 3D information from the two channels can be optimally combined to yield up to 30% improvement in localization precision relative to a single polarization channel system.

[1]  D. Chemla,et al.  Single Molecule Dynamics Studied by Polarization Modulation. , 1996, Physical review letters.

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

[3]  S. Ram,et al.  Simultaneous imaging of different focal planes in fluorescence microscopy for the study of cellular dynamics in three dimensions , 2004, IEEE Transactions on NanoBioscience.

[4]  Michael Unser,et al.  Super-resolution orientation estimation and localization of fluorescent dipoles using 3-D steerable filters. , 2009, Optics express.

[5]  Novotny,et al.  Orientational imaging of single molecules by annular illumination , 2000, Physical review letters.

[6]  Peter Török,et al.  Determination of the three-dimensional orientation of single molecules. , 2008, Optics letters.

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

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

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

[10]  Jianyong Tang,et al.  Multilayer Three-dimensional Super-resolution Imaging of Thick Biological Samples , 2008 .

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

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

[13]  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.

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

[15]  Yoav Y Schechner,et al.  Depth from diffracted rotation. , 2006, Optics letters.

[16]  George H. Patterson,et al.  A Photoactivatable GFP for Selective Photolabeling of Proteins and Cells , 2002, Science.

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

[18]  David J. Norris,et al.  SIMULTANEOUS IMAGING OF INDIVIDUAL MOLECULES ALIGNED BOTH PARALLEL AND PERPENDICULAR TO THE OPTIC AXIS , 1998 .

[19]  Rafael Piestun,et al.  High-efficiency rotating point spread functions. , 2008, Optics express.

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