Fluorescence Correlation Spectroscopy with Traveling Interference Fringe Excitation: Apparatus Using Acoustooptic Modulator Devices

A new improved fluorescence correlation spectroscopy apparatus for determining motional states of fluorescent particles, using acoustooptic modulator (AOM) devices to generate a traveling interference fringe, is presented. In this apparatus, the scheme of fluorescence correlation spectroscopy with traveling interference fringe excitation (FCSTFE) is applied. The modulated fluorescence signal from particles excited by a moving interference fringe is detected, and cosine and sine Fourier coefficients at the frequency of the traveling fringe are recorded. The autocorrelation function of the fluorescence intensity consists of terms which are characterized by the size of the excitation region and the spacing of the fringe. A lock-in detection method is adopted for extracting cosine and sine Fourier coefficients at the frequency of the traveling fringe (Fcos(t), Fsin(t)). Autocorrelation functions of Fcos(t) and Fsin(t) express the motion of the fluorescent particles. By setting the velocity of the traveling interference fringe to be much faster than the velocity of moving particles, the diffusion coefficient and velocity can be determined independently from the expression for coupled flow and diffusion. The improved apparatus has variable fringe spacings and modulation frequencies on the order of kHz, and can be used to measure the flow velocity and diffusion of small fluorescent-labeled molecules. An experiment on the determination of diffusion coefficients of dispersed fluorescent polystyrene spheres in water was demonstrated.