Fabrication of standard samples for single-molecule fluorescence imaging

A strategy for fabricating a single-molecule sample was developed to meet the requirements for a standard sample, which is characterized by uniform fluorescence intensity and uniform spatial distribution of single molecules on a glass surface. Individual chromophores of hexachlorofluorescein or Texas Red were immobilized on a glass surface by covalent bonding through molecular tethers. Fluorescence intensity and spatial distribution of individual fluorescent spots were evaluated using far-field video microscopy. Uniform fluorescence intensity was quantitatively represented by a sharp distribution in the histogram of the fluorescence photocounts. To determine the spatial distribution of fluorescent spots, uniform or not, the statistical ensemble of the number of fluorescent spots in a small area (5 x 5 and 2 x 2 mum(2)) randomly set within the full area (100 X 100 mum(2)) was collected; then, the ensemble was compared with the Poisson distribution.