Surface Preparation for Single-Molecule Fluorescence Imaging in Organic Solvents

The development of single-molecule techniques provides opportunities to investigate the properties and heterogeneities of individual molecules, which are almost impossible to be obtained in ensemble measurements. Recently, single-molecule fluorescence microscopy is being applied more and more to study chemical reactions in organic solvents. However, little has been done to optimize the surface preparation procedures for single-molecule fluorescence imaging in organic solvents. In this work, we developed a method to prepare the surface for single-molecule fluorescence imaging in organic solvents with a well-controlled surface density of chemically immobilized dye molecules and a low density of nonspecifically adsorbed impurities. We also compared the surfaces prepared by two different procedures and studied the impacts of the polarities of the solvent and the surface functionality on the quality of prepared surface. We found that higher polarities of both the solvent and the surface functionality provided better control of the surface density of chemically immobilized dyes and helped reduce the nonspecific adsorption of both dyes and fluorescent impurities in organic solvents. We further performed single-molecule fluorescence imaging in DMF and investigated the photophysical properties of dyes and fluorescent impurities, which could be used to filter out false counts in single-molecule fluorescence measurements.