Molecular dynamics of biological probes by fluorescence correlation microscopy with two-photon excitation

We report on the application of fluorescence correlation microscopy under two-photon excitation of fluorophores of biological interest: FITC-dextran (MW, from 20 to 150 kDa), green fluorescent protein (MW, 27 kDa), and fluorescein (MW, 330 Dal. Under these experimental conditions, the translational diffusion coefficients of these molecules in aqueous solutions derived from the fluorescence intensity autocorrelation function were determined for the first time and were found to be 24 x 10(-7), 8.2 x 10(-7), and 3 x 10(-7) cm(2) s(-1) for 150-kDa FITC-dextran, green fluorescent protein, and fluorescein, respectively. These results are discussed in connection with previously reported results obtained by different methods. The great sensibility of the system has been applied to single-molecule detection of the smaller fluorophore, fluorescein.