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 molecul es in solution with high classification probability based on confocal micro scopy in combination with spectrally and time-resolved fluorescence detecti on with two detectors. For efficient excitation of the labelled mononucleot ide molecules JA133-dUTP, JA169-dUTP, Cy5-dCTP, and JA242-dUTP a short-puls e diode laser emitting at 634 nm with a repetition rate of 64 MHz was appli ed. The time-resolved fluorescence signals of individual molecules were ana lyzed and identified by a maximum likelihood estimator (MLE). Scatter plots of spectrally and time-resolved fluorescence data demonstrated the existen ce of four distinct populations with symmetrical shape. The distributions o f each of the mononucleotide conjugates were determined by fitting a superp osition of two independent Gaussians. Taking only those single-molecule bur sts which contain more than 50 photon counts, three labelled mononucleotide molecules were identified in solution by spectrally and time-resolved fluo rescence spectroscopy with a probability of correct classification of appro ximate to 99%.