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 fluoresc ent probe molecules. In this method, interference between two intersec ting, coherent laser beams creates an excitation hinge pattern from wh ich fluorescence emission is monitored. Spontaneous concentration fluc tuations of fluorescent molecules within the excitation volume are det ected as excess noise on a fluorescence transient; concentration fluct uations are driven primarily by diffusion of these molecules between i nterference fringes although contributions from photobleaching and dif fusion over the entire pattern dimensions can also be observed. Autoco rrelation of the fluorescence transient allows analysis of the tempora l characteristics of the fluctuations, which were used to determine so lution diffusion coefficients; the method was applied to study the dif fusion of Rhodamine 6G (R6G) in water/methanol solutions containing ad ded electrolyte and in pure ethanol. The method can be used to charact erize the diffusive transport of fluorescently labeled species, which is an important issue in designing small-volume detection experiments.