Time-resolved photon counting allows for new temporal and spatial insights into the nanoworld

The time-correlated single photon counting (TCSPC) technique combined with clock oscillator set by the pulsed laser provides a precise measurement of the arrival time of the detected photons with picosecond resolution for a time-scale of hours. If TCSPC is combined with other experimental techniques such as optical spectroscopy and mechanical manipulation, it is possible to coincide the detected fluorescence signal with the changes of the sample properties. High temporal resolution achieved in TCSPC (down to ps) allows us to monitor fast mechanical processes in single molecules. Here we present recent developments in fluorescence correlation spectroscopy (FCS) as well as the combination of TCSPC with optical scanning microscopy and mechanical manipulation by means of an atomic-force microscope (AFM).

[1]  M. Konrad,et al.  Molecular Dynamics Simulation of DNA Stretching Is Consistent with the Tension Observed for Extension and Strand Separation and Predicts a Novel Ladder Structure , 1996 .

[2]  Shimon Weiss,et al.  Ratiometric measurement and identification of single diffusing molecules , 1999 .

[3]  Ralf Kühnemuth,et al.  Principles of single molecule multiparameter fluorescence spectroscopy , 2001 .

[4]  H. Gaub,et al.  Force spectroscopy with single bio-molecules. , 2000, Current opinion in chemical biology.

[5]  R. S. Goody,et al.  Multiparameter single-molecule fluorescence spectroscopy reveals heterogeneity of HIV-1 reverse transcriptase:primer/template complexes , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[6]  H Peter Lu.  Probing single-molecule protein conformational dynamics. , 2005, Accounts of chemical research.

[7]  Jerker Widengren,et al.  Single-molecule detection and identification of multiple species by multiparameter fluorescence detection. , 2006, Analytical chemistry.

[8]  Gerber,et al.  Atomic Force Microscope , 2020, Definitions.

[9]  Ü. Mets,et al.  Antibunching and Rotational Diffusion in FCS , 2001 .

[10]  Christian Eggeling,et al.  Single-Molecule Identification of Coumarin-120 by Time-Resolved Fluorescence Detection: Comparison of One- and Two-Photon Excitation in Solution , 1997 .

[11]  Hendrik Dietz,et al.  Protein structure by mechanical triangulation , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[12]  Bernard Valeur,et al.  New trends in fluorescence spectroscopy : applications to chemical and life sciences , 2001 .

[13]  Luis Moroder,et al.  Single-Molecule Optomechanical Cycle , 2002, Science.

[14]  C. Seidel,et al.  Full correlation from picoseconds to seconds by time-resolved and time-correlated single photon detection , 2005 .

[15]  Jerker Widengren,et al.  Photophysical Aspects of FCS Measurements , 2001 .

[16]  W. E. Moerner,et al.  A Dozen Years of Single-Molecule Spectroscopy in Physics, Chemistry, and Biophysics , 2002 .

[17]  C. Bustamante,et al.  Ten years of tension: single-molecule DNA mechanics , 2003, Nature.

[18]  Christoph Bräuchle,et al.  Simultaneous AFM manipulation and fluorescence imaging of single DNA strands. , 2005, Chemphyschem : a European journal of chemical physics and physical chemistry.

[19]  Wolfgang M. Heckl,et al.  Procedures in scanning probe microscopy , 1998 .

[20]  Markus Sauer,et al.  Branching Out of Single‐Molecule Fluorescence Spectroscopy: Challenges for Chemistry and Influence on Biology , 2005 .

[21]  J P McNamee,et al.  Photophysical Properties of Fluorescent DNA-dyes Bound to Single- and Double-stranded DNA in Aqueous Buffered Solution¶ , 2001, Photochemistry and photobiology.

[22]  R Lavery,et al.  Modelling extreme stretching of DNA. , 1996, Nucleic acids research.

[23]  Klaus Schaetzel,et al.  Noise on multiple-tau photon correlation data , 1991, Photonics West - Lasers and Applications in Science and Engineering.

[24]  T. Yanagida,et al.  Imaging And Nano‐Manipulation Of Single Actomyosin Motors At Work , 2000, Clinical and experimental pharmacology & physiology.

[25]  Helmut Grubmüller,et al.  Force spectroscopy of single biomolecules. , 2002, Chemphyschem : a European journal of chemical physics and physical chemistry.

[26]  H. Grubmüller,et al.  Single-molecule fluorescence resonance energy transfer reveals a dynamic equilibrium between closed and open conformations of syntaxin 1 , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[27]  A Volkmer,et al.  Data registration and selective single-molecule analysis using multi-parameter fluorescence detection. , 2001, Journal of biotechnology.

[28]  H. Masuhara,et al.  Time-Dependent Fluorescence Depolarization Analysis in Three-Dimensional Microspectroscopy , 1995 .

[29]  Michel Orrit,et al.  Ten Years of Single-Molecule Spectroscopy. , 2000, The journal of physical chemistry. A.

[30]  W. Becker.  Advanced Time-Correlated Single Photon Counting Techniques , 2005 .

[31]  M. Orrit,et al.  Single pentacene molecules detected by fluorescence excitation in a p-terphenyl crystal. , 1990, Physical review letters.

[32]  Enrico Gratton,et al.  Measuring fast dynamics in solutions and cells with a laser scanning microscope. , 2005, Biophysical journal.

[33]  Christian Eggeling,et al.  Detection and characterization of single molecules in aqueous solution , 1996 .

[34]  C. Seidel,et al.  Monitoring conformational dynamics of a single molecule by selective fluorescence spectroscopy. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[35]  Elisha Haas,et al.  The study of protein folding and dynamics by determination of intramolecular distance distributions and their fluctuations using ensemble and single-molecule FRET measurements. , 2005, Chemphyschem : a European journal of chemical physics and physical chemistry.

[36]  Janshoff,et al.  Force Spectroscopy of Molecular Systems-Single Molecule Spectroscopy of Polymers and Biomolecules. , 2000, Angewandte Chemie.