Time-resolved detection and identification of single analyte molecules in microcapillaries by time-correlated single-photon counting (TCSPC)

A PC plug-in card for on-line time resolved fluorescence detection of single dye molecules based on a new time-correlated single photon counting (TCSPC) module is described. The module contains all electronic components constant fraction discriminators (CFDs), time-to-amplitude converter (TAC), analog-to-digital converter (ADC), multichannel analyzer (MCA timers) on board required for TCSPC. A fast TAC design in combination with a fast flash ADC and an error-correcting ADC/MCA principle results in a maximum count rate of 8 MHz (dead time 125 ns). A dual memory architecture allows for unlimited recording of decay curves with collection times down to 150 μs without time gaps between subsequent recordings. Applying a short-pulse diode laser emitting at 640 nm with a repetition rate of 60 MHz in combination with a confocal microscope, we studied bursts of fluorescence photons from individual dye labeled mononucleotide molecules (Cy5-dCTP) in a cone shaped microcapillary with an inner diameter of 0.5 μm at the...

[1]  F. Regnier,et al.  Capillary electrophoretic separations of proteins using nonionic surfactant coatings. , 1991, Analytical chemistry.

[2]  M. Eigen,et al.  Sorting single molecules: application to diagnostics and evolutionary biotechnology. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[3]  J H Jett,et al.  Detection of single molecules of phycoerythrin in hydrodynamically focused flows by laser-induced fluorescence. , 1987, Analytical chemistry.

[4]  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.

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

[6]  Steven A. Soper,et al.  Detection of single fluorescent molecules , 1990 .

[7]  R. Hochstrasser,et al.  Nonexponential kinetics of a single tRNAPhe molecule under physiological conditions. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

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

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

[10]  M. Sauer,et al.  Detection and identification of individual antigen molecules in human serum with pulsed semiconductor lasers , 1997 .

[11]  James H. Jett,et al.  An Approach To Single Molecule Detection By Laser-Induced Fluorescence , 1983, Optics & Photonics.

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

[13]  Steven A. Soper,et al.  Detection and identification of single molecules in solution , 1992 .

[14]  A. Castro,et al.  Single-molecule detection: applications to ultrasensitive biochemical analysis. , 1995, Applied optics.

[15]  R A Mathies,et al.  Single-molecule fluorescence detection: autocorrelation criterion and experimental realization with phycoerythrin. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[16]  R N Zare,et al.  Probing individual molecules with confocal fluorescence microscopy. , 1994, Science.

[17]  Jürgen Wolfrum,et al.  Single-molecule counting and identification in a microcapillary , 1998 .

[18]  M. Eigen,et al.  Techniques for single molecule sequencing , 1997, Bioimaging.

[19]  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.

[20]  W. P. Ambrose,et al.  A maximum likelihood estimator to distinguish single molecules by their fluorescence decays , 1997 .

[21]  W. Whitten,et al.  High-efficiency molecular counting in solution:  single-molecule detection in electrodynamically focused microdroplet streams. , 1997, Analytical chemistry.

[22]  Richard A. Keller,et al.  Single-Molecule Detection in Liquids by Laser-Induced Fluorescence , 1996 .

[23]  G. Deltau,et al.  Time-resolved identification of single molecules in solution with a pulsed semiconductor diode laser , 1996 .

[24]  Shuming Nie,et al.  Confinement and Detection of Single Molecules in Submicrometer Channels , 1997 .