Identification of single fluorescently labelled mononucleotide molecules in solution by spectrally resolved time-correlated single-photon counting

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%.