High-resolution colocalization of single molecules within the resolution gap of far-field microscopy.

To obtain detailed information about the three-dimensional (3D) organization of small biomolecular assemblies with a size of less than 100 nanometers, advanced techniques are required that enable the determination of absolute 3D positions and distances between individual fluorophores well below the resolution limit of conventional light microscopy. We show how spectrally resolved fluorescence lifetime imaging microscopy (SFLIM) can provide significant contributions and allow us to determine distances between conventional individual fluorophores (Bodipy 630/650 and Cy5.5) that are less than 20 nm apart. We take advantage of fluorescent dyes (here Cy5.5 and Bodipy 630/650) that can be efficiently excited by a single pulsed diode laser emitting at 635 nm but differ in their fluorescence lifetime and emission maxima. The potential of the method for ultrahigh colocalization studies is demonstrated by measuring the end-to-end distance between single fluorophores separated by double-stranded DNA of various lengths. Combining SFLIM with polarization-modulated excitation allows us to obtain, simultaneously, information about the relative orientation of fluorophores. Furthermore, we show that the environment-dependent photophysics of conventional fluorophores, that is, photostability, blinking pattern, and the tendency to enter irreversible nonfluorescent states, sets certain limitations to their in vitro and in vivo applications.

[1]  D. Jackson,et al.  Regional specialization in human nuclei: visualization of discrete sites of transcription by RNA polymerase III , 1999, The EMBO journal.

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

[3]  Stefan W. Hell,et al.  Laser-diode-stimulated emission depletion microscopy , 2003 .

[4]  Richard A. Keller,et al.  Analysis of fluorescence lifetime data for single rhodamine molecules in flowing sample streams , 1994 .

[5]  X Michalet,et al.  Ultrahigh-resolution colocalization of spectrally separable point-like fluorescent probes. , 2001, Methods.

[6]  Kenneth D. Weston,et al.  Orientation Imaging and Reorientation Dynamics of Single Dye Molecules , 2001 .

[7]  W. Webb,et al.  Precise nanometer localization analysis for individual fluorescent probes. , 2002, Biophysical journal.

[8]  Thomas Heinlein,et al.  A Single-Molecule Sensitive DNA Hairpin System Based on Intramolecular Electron Transfer , 2003 .

[9]  H Schindler,et al.  Imaging of single molecule diffusion. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[10]  P. Cook.  The organization of replication and transcription. , 1999, Science.

[11]  Rainer Heintzmann,et al.  High-resolution colocalization of single dye molecules by fluorescence lifetime imaging microscopy. , 2002, Analytical chemistry.

[12]  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.

[13]  M. Sauer,et al.  Photophysical Dynamics of Single Molecules Studied by Spectrally-Resolved Fluorescence Lifetime Imaging Microscopy (SFLIM) , 2001 .

[14]  A. Lamond,et al.  Structure and function in the nucleus. , 1998, Science.

[15]  Jürgen Köhler,et al.  Far-field fluorescence microscopy beyond the diffraction limit , 1999 .

[16]  Paul R. Selvin,et al.  The renaissance of fluorescence resonance energy transfer , 2000, Nature Structural Biology.

[17]  W. Moerner,et al.  Illuminating single molecules in condensed matter. , 1999, Science.

[18]  C Cremer,et al.  Three‐dimensional spectral precision distance microscopy of chromatin nanostructures after triple‐colour DNA labelling: a study of the BCR region on chromosome 22 and the Philadelphia chromosome , 2000, Journal of microscopy.

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

[20]  Christoph Cremer,et al.  Superresolution size determination in fluorescence microscopy: A comparison between spatially modulated illumination and confocal laser scanning microscopy , 2004 .

[21]  D. Spector,et al.  Macromolecular domains within the cell nucleus. , 1993, Annual review of cell biology.

[22]  Radial Distribution Function of Semiflexible Polymers. , 1996, Physical review letters.

[23]  Mircea Cotlet,et al.  Antibunching in the emission of a single tetrachromophoric dendritic system. , 2002, Journal of the American Chemical Society.

[24]  H. Leonhardt,et al.  Reversal of terminal differentiation and control of DNA replication: Cyclin A and cdk2 specifically localize at subnuclear sites of DNA replication , 1993, Cell.

[25]  R. Vale,et al.  Kinesin Walks Hand-Over-Hand , 2004, Science.

[26]  Shimon Weiss,et al.  Measuring conformational dynamics of biomolecules by single molecule fluorescence spectroscopy , 2000, Nature Structural Biology.

[27]  X. Xie,et al.  Optical studies of single molecules at room temperature. , 1998, Annual review of physical chemistry.

[28]  T. Kues,et al.  Imaging and tracking of single GFP molecules in solution. , 2000, Biophysical journal.

[29]  Ulrich Kubitscheck.  Single Protein Molecules Visualized and Tracked in the Interior of Eukaryotic Cells , 2002 .

[30]  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.

[31]  S. Weiss.  Fluorescence spectroscopy of single biomolecules. , 1999, Science.

[32]  M. Sauer,et al.  Fluorescence resonance energy transfer (FRET) and competing processes in donor-acceptor substituted DNA strands: a comparative study of ensemble and single-molecule data. , 2002, Journal of biotechnology.

[33]  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.

[34]  E. Manders,et al.  Chromatic shift in multicolour confocal microscopy , 1997 .

[35]  R. Zare,et al.  Optical detection of single molecules. , 1997, Annual review of biophysics and biomolecular structure.

[36]  E. Siggia,et al.  Entropic elasticity of lambda-phage DNA. , 1994, Science.

[37]  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.

[38]  J. Spudich,et al.  Single molecule high-resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[39]  W. Moerner,et al.  Three-Dimensional Imaging of Single Molecules Solvated in Pores of Poly(acrylamide) Gels , 1996, Science.