Molecular Orientation and Its Influence on Autocorrelation Amplitudes in Single-Molecule Imaging Experiments

The concentration dependence of autocorrelation amplitudes obtained from orientationally fixed single molecules is explored using theory, simulation, and experimental results. Autocorrelation amplitudes obtained under such circumstances are shown to be approximately 2-fold larger than predicted in previous studies (Koppel, D. E. Phys. Rev. A 1974, 10, 1938-1945 and citing references), which frequently assume polarization-independent excitation and detection. A detailed derivation of the autocorrelation amplitude expected under conditions frequently employed in single-molecule experiments is given. Simulated and experimental single-molecule image data obtained from samples incorporating fixed single molecules are used to verify the correctness of the model. These results are compared to both simulated and experimental time transient data in which the molecules exhibit predominantly fast rotational reorientation and to which previously reported models apply. The experimental results employed are obtained from dye-doped mesoporous silica thin films studied at different levels of hydration. The theory and results obtained are of importance to the determination of molecular concentrations from single-molecule image and time transient autocorrelation data, in situations where the molecules exhibit permanent or reversible adsorption at fixed orientations in or on thin-film materials.