Confocal Fluorescence Lifetime Imaging Microscopy (FLIM) at the Single Molecule Level

We report on confocal fluorescence lifetime imaging microscopy (CFLIM) of single dye molecules adsorbed on glass surface. Applying a short-pulse diode laser emitting at 635 nm with a repetition rate of 64 MHz we studied the time-resolved identification of individual carbocyanine and oxazine dyes via their characteristic fluorescence lifetimes of 2.06±0.37 ns (Cy5) and 3.89±0.91 ns (JA242). Fluctuations in fluorescence intensity and lifetime of individual adsorbed molecules were investigated with millisecond time resolution. These jumps exhibit short off-states τoff of 0.5 ms which can be ascribed to the triplet state lifetime under dry conditions with an intersystem crossing yield YISC of ∼0.2 %. Besides triplet states, other quantum jumps into longer lived states (several milliseconds) with lower transition probability were observed. The correlation of rotational and spectral jumps of single and coupled fluorophores with changes in the observed fluorescence lifetime are discussed.

[1]  M. Sauer,et al.  Single molecule DNA sequencing in submicrometer channels: state of the art and future prospects. , 2001, Journal of biotechnology.

[2]  W. P. Ambrose,et al.  Single molecule fluorescence spectroscopy at ambient temperature. , 1999, Chemical reviews.

[3]  N. F. van Hulst,et al.  Time-varying triplet state lifetimes of single molecules , 1999 .

[4]  E. Peterman,et al.  Optical methods for exploring dynamics of single copies of green fluorescent protein. , 1999, Cytometry.

[5]  M Dahan,et al.  Single-pair fluorescence resonance energy transfer on freely diffusing molecules: observation of Förster distance dependence and subpopulations. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

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

[7]  Jürgen Wolfrum,et al.  Time-resolved detection and identification of single analyte molecules in microcapillaries by time-correlated single-photon counting (TCSPC) , 1999 .

[8]  W. B. Caldwell,et al.  Single-molecule fluorescence spectroscopy of enzyme conformational dynamics and cleavage mechanism. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[9]  Andreas Volkmer,et al.  Identification of Single Molecules in Aqueous Solution by Time-Resolved Fluorescence Anisotropy , 1999 .

[10]  Liming Ying,et al.  Fluorescence Spectroscopy, Exciton Dynamics and Photochemistry of Single Allophycocyanin Trimers , 1998 .

[11]  Kenneth D. Weston,et al.  Millisecond Intensity Fluctuations of Single Molecules at Room Temperature , 1998 .

[12]  B. Steipe,et al.  Confocal microscopy of single molecules of the green fluorescent protein , 1998 .

[13]  Jürgen Wolfrum,et al.  Time‐resolved identification of individual mononucleotide molecules in aqueous solution with pulsed semiconductor lasers , 1998 .

[14]  Markus Sauer,et al.  Dynamics of the electron transfer reaction between an oxazine dye and DNA oligonucleotides monitored on the single-molecule level , 1998 .

[15]  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.

[16]  E. Yeung,et al.  Monitoring the Reactions of Single Enzyme Molecules and Single Metal Ions , 1997 .

[17]  Paul F. Barbara,et al.  Discrete Intensity Jumps and Intramolecular Electronic Energy Transfer in the Spectroscopy of Single Conjugated Polymer Molecules , 1997 .

[18]  D. Chemla,et al.  Quantum jumps of single molecules at room temperature , 1997 .

[19]  E. Yeung,et al.  Direct Measurement of Single-Molecule Diffusion and Photodecomposition in Free Solution , 1997, Science.

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

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

[22]  James H. Jett,et al.  Single-Molecule Fluorescence Analysis in Solution , 1996 .

[23]  R. Rigler,et al.  Conformational transitions monitored for single molecules in solution. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[24]  D. F. Ogletree,et al.  Probing the interaction between single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor , 1996, Summaries of Papers Presented at the Quantum Electronics and Laser Science Conference.

[25]  Louis E. Brus,et al.  Imaging and Time-Resolved Spectroscopy of Single Molecules at an Interface , 1996, Science.

[26]  W. P. Ambrose,et al.  Alterations of Single Molecule Fluorescence Lifetimes in Near-Field Optical Microscopy , 1994, Science.

[27]  Charles W. Wilkerson,et al.  Bias and precision in the estimation of exponential decay parameters from sparse data , 1993 .

[28]  Lewenstein,et al.  Cooperative quantum jumps with two atoms. , 1987, Physical review letters.

[29]  M. Sauer,et al.  Detection and identification of single dye labeled mononucleotide molecules released from an optical fiber in a microcapillary: First steps towards a new single molecule DNA sequencing technique , 1999 .

[30]  R. Menzel,et al.  Influence of chemical substitution and electronic effects on the triplet state kinetics of xanthene dyes , 1999 .

[31]  H. Peter Lu,et al.  Single-molecule spectral fluctuations at room temperature , 1997, Nature.

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

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

[34]  L. Stryer.  Fluorescence energy transfer as a spectroscopic ruler. , 1978, Annual review of biochemistry.

[35]  Th. Förster.  Zwischenmolekulare Energiewanderung und Fluoreszenz , 1948 .