%0 Journal Article %1 springerlink:10.1007/s003400000405 %A Herten, D.P. %A Tinnefeld, P. %A Sauer, M. %D 2000 %I Springer Berlin / Heidelberg %J Applied Physics B: Lasers and Optics %K sauer %P 765-771 %T Identification of single fluorescently labelled mononucleotide molecules in solution by spectrally resolved time-correlated single-photon counting %U http://dx.doi.org/10.1007/s003400000405 %V 71 %X We describe a method to identify single dye-labelled mononucleotide molecules in solution with high classification probability based on confocal microscopy in combination with spectrally and time-resolved fluorescence detection with two detectors. For efficient excitation of the labelled mononucleotide molecules JA133-dUTP, JA169-dUTP, Cy5-dCTP, and JA242-dUTP a short-pulse diode laser emitting at 634 nm with a repetition rate of 64 MHz was applied. The time-resolved fluorescence signals of individual molecules were analyzed and identified by a maximum likelihood estimator (MLE). Scatter plots of spectrally and time-resolved fluorescence data demonstrated the existence of four distinct populations with symmetrical shape. The distributions of each of the mononucleotide conjugates were determined by fitting a superposition of two independent Gaussians. Taking only those single-molecule bursts which contain more than 50 photon counts, three labelled mononucleotide molecules were identified in solution by spectrally and time-resolved fluorescence spectroscopy with a probability of correct classification of ≈99%.

@article{springerlink:10.1007/s003400000405, abstract = {We describe a method to identify single dye-labelled mononucleotide molecules in solution with high classification probability based on confocal microscopy in combination with spectrally and time-resolved fluorescence detection with two detectors. For efficient excitation of the labelled mononucleotide molecules JA133-dUTP, JA169-dUTP, Cy5-dCTP, and JA242-dUTP a short-pulse diode laser emitting at 634 nm with a repetition rate of 64 MHz was applied. The time-resolved fluorescence signals of individual molecules were analyzed and identified by a maximum likelihood estimator (MLE). Scatter plots of spectrally and time-resolved fluorescence data demonstrated the existence of four distinct populations with symmetrical shape. The distributions of each of the mononucleotide conjugates were determined by fitting a superposition of two independent Gaussians. Taking only those single-molecule bursts which contain more than 50 photon counts, three labelled mononucleotide molecules were identified in solution by spectrally and time-resolved fluorescence spectroscopy with a probability of correct classification of ≈99%.}, added-at = {2011-03-15T08:52:28.000+0100}, affiliation = {Physikalisch-Chemisches Institut, Universität Heidelberg, Im Neuenheimer Feld 253, 69120 Heidelberg, Germany (Fax: +49-6221/544-255, E-mail: sauer@urz.uni-heidelberg.de) DE}, author = {Herten, D.P. and Tinnefeld, P. and Sauer, M.}, biburl = {https://www.bibsonomy.org/bibtex/26340810b94745a8573eac724a5c85bf7/reichert}, interhash = {22fb5e5fc648cf41ec0828bb656f22de}, intrahash = {6340810b94745a8573eac724a5c85bf7}, issn = {0946-2171}, issue = {5}, journal = {Applied Physics B: Lasers and Optics}, keyword = {Physics and Astronomy}, keywords = {sauer}, note = {10.1007/s003400000405}, pages = {765-771}, publisher = {Springer Berlin / Heidelberg}, timestamp = {2011-03-15T08:52:29.000+0100}, title = {Identification of single fluorescently labelled mononucleotide molecules in solution by spectrally resolved time-correlated single-photon counting}, url = {http://dx.doi.org/10.1007/s003400000405}, volume = 71, year = 2000 }