Quantitative dosing of surfaces with fluorescent molecules: characterization of fractional monolayer coverages by counting single molecules.

Quantitative deposition of dye molecules onto a substrate has been achieved at very low surface concentrations, in the range of 5 x 10(-8) - 1 x 10(-6) monolayer, using the technique of controlled substrate withdrawal from solution. These small surface populations were determined with high (>96%) efficiency by single-molecule counting using an epi-illumination, fluorescence microscope with charge-coupled device detector. The fluorescence imaging resolution (3sigma) is 0.78 microm; over a uniform excitation area of 67 x 67 microm2, a large number (>7,500) of spatially resolved channels are available for counting individual molecules. At low coverages, the number density of fluorescence spots on the surface agrees with the expected surface concentration of molecules, based on the concentration of dye in solution and the solution film thickness predicted from theory. When the surface density of molecules is high enough that fluorescence spot overlap is likely to occur within the optical resolution of the instrument, the observed fewer number of spots can be corrected for overlap through a site occupation model based on Poisson statistics.