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Sensitivity of second harmonic generation from styryl dyes to transmembrane potential
Andrew C Millard1, Lei Jin, Mei-De Wei
1Department of Physiology and Center for Biomedical Imaging Technology, University of Connecticut Health Center, Farmington, Connecticut 06030-1507, USA.
Biophysical Journal
|January 30, 2004
Summary
Researchers developed advanced nonlinear imaging techniques to measure cell membrane potential. Second harmonic generation (SHG) imaging with styryl dyes offers a fourfold improvement in voltage sensitivity compared to traditional methods.
Area of Science:
- Biophysics
- Cell Biology
- Optical Imaging
Background:
- Accurate measurement of transmembrane potential is crucial for understanding cellular function.
- Existing voltage-sensitive dyes often have limitations in sensitivity and photostability.
- Nonlinear optical microscopy offers novel approaches for cellular analysis.
Purpose of the Study:
- To evaluate the voltage sensitivity of second harmonic generation (SHG) using ANEP-chromophore styryl dyes.
- To investigate the influence of excitation wavelength and dye structure on SHG voltage sensitivity.
- To compare SHG voltage sensitivity with conventional one-photon fluorescence methods.
Main Methods:
- Simultaneous nonlinear imaging (SHG and two-photon excitation fluorescence) and voltage clamping of living cells.
- Systematic variation of excitation wavelength and styryl dye structure.
- Quantification of SHG signal changes in response to transmembrane potential shifts.
Main Results:
- Achieved SHG voltage sensitivities up to 43% per 100 mV.
- Demonstrated a sensitivity improvement of over fourfold compared to one-photon fluorescence.
- Observed excitation wavelength dependence consistent with two-photon resonance.
- Identified significant impact of dye structure on voltage sensitivity.
Conclusions:
- SHG imaging with specifically designed styryl dyes provides a highly sensitive method for monitoring membrane potential.
- The observed wavelength and structural dependencies offer a pathway for optimizing voltage-sensitive dye design.
- This technique advances the capability for real-time electrophysiological studies in living cells.