Characterization of particle motions by fluorescence correlation spectroscopy with traveling interference fringe excitation

A new improved fluorescence correlation spectroscopy method for determining motional states of particles, fluorescence correlation spectroscopy with traveling interference fringe excitation (FCSTFE), is presented. In this method the modulated fluorescence signal from particles excited by an interference fringe moving at constant velocity is detected, and cosine and sine Fourier coefficients at the frequency of the traveling fringe are recorded. Autocorrelation functions of Fourier coefficients and the square of amplitude were found to express the motion of the fluorescent particles and to correspond to that of laser light scattering experiment in homodyne mode. The apparatus utilizing a photon counting system was constructed and the performance of the system in measuring diffusion coefficients of fluorescent polystyrene spheres suspended in water and ethylene glycol mixed solvents was demonstrated. The FCSTFE experiments using biological membranes are expected to yield similar information to that obtained from ideal laser light scattering experiments which are difficult to perform because of the complexity in data analysis due to stray light and multiple scattered light from the samples. The new method is also superior in avoiding severe problems, such as the damage in samples caused by the intense bleaching light, which may occur during the fluorescence photobleaching recovery.© (1996) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.