ANALYSIS OF SINGLE FLUORESCENT MOLECULES TRAPPED INSIDE SMALL LIPID VESICLES

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A rationale for improvement of the response (fractional change in fluorescence, (F/F) of a voltage-sensitive dye has been tested. Improvement of current voltage-sensitive dyes which currently show (F/F of 10% at best, would be of great interest to neuroscientists. The two-photon action cross-section of Di-8-ANEPPS, a bright, photostable, relatively non-cytotoxic dye was measured relative to Rhodamine B in MeOH from 800-1000 nm. A two-photon exctiation wavelength was chosen to maximize the magnitude and slope of the cross-section with respect to wavelength. Hela cells were loaded with the dye and imaged before and after K+ depolarization to quantify (F/F for a large change in membrane potential. No conclusive improvement was observed using two-photon excitation. Further investigation of the same molecule using patch-clamp techniques and different excitation wavelengths is underway.