Determination of the diffusion coefficient of dye in solution at single molecule level

We have analyzed the width distributions of fluorescence bursts emitted from single dye in solution. Here we used the confocal microscope combined with a short-pulsed diode laser providing an excitation wavelength of 638 nm and a repetition rate of 17 MHz. Under the experimental condition that the transit times of dye molecules through a probe space were shorter than the average photodestruction time, the data obtained using a multichannel scaler showed a linear trend between the photon counts and the width of fluorescence bursts. From the width distributions of burst, the translational diffusion coefficient of JA22 in ethylene glycol was determined.