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Sensitivity of Second Harmonic Generation from Styryl Dyes to Transmembrane Potential

Andrew C. Millard ^*, Lei Jin ^*, Mei-de Wei ^*, Joseph P. Wuskell ^*, Aaron Lewis  and Leslie M. Loew ^*

^* Department of Physiology and Center for Biomedical Imaging Technology, University of Connecticut Health Center, Farmington, Connecticut; and Division of Applied Physics, Hebrew University, Jerusalem, Israel

Correspondence: Address reprint requests to Leslie M. Loew, Center for Biomedical Imaging Technology, University of Connecticut Health Center, 263 Farmington Ave. MC-1507, Farmington, CT 06030-1507. Tel.: 860-679-3568; Fax: 860-679-1039; E-mail: les{at}volt.uchc.edu.

In this article we present results from the simultaneous nonlinear (second harmonic generation and two-photon excitation fluorescence) imaging and voltage clamping of living cells. Specifically, we determine the sensitivity to transmembrane potential of second harmonic generation by ANEP-chromophore styryl dyes as a function of excitation wavelength and dye structure. We have measured second harmonic sensitivities of up to 43% per 100 mV, more than a factor of four better than the nominal voltage sensitivity of the dyes under "one-photon" fluorescence. We find a dependence of voltage sensitivity on excitation wavelength that is consistent with a two-photon resonance, and there is a significant dependence of voltage sensitivity on the structure of the nonchromophore portion of the dyes.

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