Fluorescence correlation spectroscopy with patterned photoexcitation for measuring solution diffusion coefficients of robust fluorophores

Patterned fluorescence correlation spectroscopy is developed as a new technique for measuring diffusion coefficients of photostable fluorescent probe molecules. In this method, interference between two intersecting, coherent laser beams creates an excitation hinge pattern from which fluorescence emission is monitored. Spontaneous concentration fluctuations of fluorescent molecules within the excitation volume are detected as excess noise on a fluorescence transient; concentration fluctuations are driven primarily by diffusion of these molecules between interference fringes although contributions from photobleaching and diffusion over the entire pattern dimensions can also be observed. Autocorrelation of the fluorescence transient allows analysis of the temporal characteristics of the fluctuations, which were used to determine solution diffusion coefficients; the method was applied to study the diffusion of Rhodamine 6G (R6G) in water/methanol solutions containing added electrolyte and in pure ethanol. The method can be used to characterize the diffusive transport of fluorescently labeled species, which is an important issue in designing small-volume detection experiments.