Fluorescence Correlation Spectroscopy Method Research Articles

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 described. With 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 [Fcos(t), Fsin(t)] are recorded. Autocorrelation functions of Fcos(t), Fsin(t), and the square of amplitude [A2(t)=Fcos(t)2+Fsin(t)2] were found to express the motion of the fluorescent particles. The apparatus utilizing a photon counting system was constructed, and the performance of the system was demonstrated by the determination of diffusion coefficients of dispersed fluorescent polystyrene spheres. Features of FCSTFE in particle dynamics evaluation are discussed.

Proposal of a Simple Method of Fluorescence Correlation Spectroscopy for Measuring the Direction and Magnitude of a Flow of Fluorophores

A new simple method to determine the direction and magnitude of flow of a fluorescence-labeled macromolecule in a solution or tissue is proposed, in which the fluorescence correlation function due to fluctuations of fluorescence intensity excited by a rectangular cross-section laser light beam or an ellipsoidal Gaussian laser light beam is measured. By this method, the contribution of translational Brownian motion of the fluorophore, if any, to the fluorescence correlation function can also be estimated as well as that of flow motion.

Motion of myosin fragments during actin‐activated ATPase: fluorescence correlation spectroscopy study

AbstractThe rates of the translational motion of myosin fragments, heavy meromyosin (HMM), and heavy meromyosin subfragment‐1 (HMM S‐1) were measured during actin‐activated ATPase reaction by the method of fluorescence correlation spectroscopy. This technique monitors the random fluctuations in the concentration of fluorescent molecules in an open volume which result from the translational diffusion of the molecular species under observation. The statistical behavior of the fluctuations is represented in the form of the autocorrelation function, which is related to the translational diffusion coefficient of the fluorescent molecules. The translational motion of fluorescently labeled myosin fragments was progressively slowed down after additions of increasing amounts of actin in the presence of excess MgATP. When these results are interpreted according to a simple binding scheme, the extent of the retardation can be used to obtain the apparent association constant for binding of S‐1 and HMM to actin in the presence of MgATP. In 0.1M KCl and at 23°C, the apparent association constants were determined as KappHMM = 2.2 × 104M−1 and KappS‐1 = 8.8 × 103 for HMM and S‐1, respectively.