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 (5×5 and 2×2 µm 2 ) randomly set within the full area (100×100 µm 2 ) was collected; then, the ensemble was compared with the Poisson distribution.