By using the single molecule detection (SMD) technique based on laser scanning fluorescence microscopy, we have developed a method to count the number of labeled fluorophores in a single biomolecule. The number of labeled fluorophores in an individual molecule was measured by counting the number of discrete level jumps in the fluorescence-intensity transients, or Gaussian fitting of the fluorescence-intensity histogram. The instabilities of intensity transients due to humid environments of biomolecules were minimized by optimizing the matrix materials and sample-preparation condition and adding a strong triplet quencher, AET (2-aminoethanethiol). After validating the counting method and estimating the counting uncertainties through observations of a well-defined number of fluorophores, the distribution of the number of incorporated fluorophores in a DNA strand was observed. The advantages in biological analysis by using a single-molecule fluorescence technique have been considered.
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