The Journal of Membrane Biology

, Volume 208, Issue 2, pp 103–111

Wavelength- and Time-Dependence of Potentiometric Non-linear Optical Signals from Styryl Dyes

  • A.C. Millard
  • L. Jin
  • J.P. Wuskell
  • D.M. Boudreau
  • A. Lewis
  • L.M. Loew
Article

DOI: 10.1007/s00232-005-0823-y

Cite this article as:
Millard, A., Jin, L., Wuskell, J. et al. J Membrane Biol (2005) 208: 103. doi:10.1007/s00232-005-0823-y

Abstract

Second harmonic generation (SHG) imaging microscopy is an important emerging technique for biological research, complementing existing one- and two-photon fluorescence (2PF) methods. A non-linear phenomenon employing light from mode-locked Ti:sapphire or fiber-based lasers, SHG results in intrinsic optical sectioning without the need for a confocal aperture. Furthermore, as a second-order process SHG is confined to loci lacking a center of symmetry, a constraint that is readily satisfied by lipid membranes with only one leaflet stained by a dye. Of particular interest is “resonance-enhanced” SHG from styryl dyes in cellular membranes and the possibility that SHG is sensitive to transmembrane potential. We have previously confirmed this, using simultaneous voltage-clamping and non-linear imaging of cells to find that SHG is up to four times more sensitive to potential than fluorescence. In this work, we have extended these results in two directions. First, with a range of wavelengths available from a mode-locked Ti:sapphire laser and a fiber-based laser, we have more fully investigated SHG and 2PF voltage-sensitivity from ANEP and ASTAP chromophores, obtaining SHG sensitivity spectra that are consistent with resonance enhancements. Second, we have modified our system to coordinate the application of voltage-clamp steps with non-linear image acquisition to more precisely characterize the time dependence of SHG and 2PF voltage sensitivity, finding that, at least for some dyes, SHG responds more slowly than fluorescence to changes in transmembrane potential.

Keywords

Non-linear imagingResonance enhancementPatch clampVoltage sensitivityTransmembrane potentialFluorescence

Copyright information

© Springer Science+Business Media, Inc. 2006

Authors and Affiliations

  • A.C. Millard
    • 1
  • L. Jin
    • 1
    • 2
  • J.P. Wuskell
    • 1
  • D.M. Boudreau
    • 1
    • 3
  • A. Lewis
    • 1
  • L.M. Loew
    • 1
  1. 1.Center for Cell Analysis and ModelingUniversity of Connecticut Health CenterFarmingtonUSA
  2. 2.Department of Biomedical and Health Information SciencesUniversity of Illinois at ChicagoChicagoUSA
  3. 3.R.T. Vanderbilt Company, Inc.NorwalkUSA