title: Second-harmonic generation imaging of membrane potential with retinal analogues.
abstract: Second-harmonic generation (SHG) by membrane-incorporated probes is a nonlinear optical signal that is voltage-sensitive and the basis of a sensitive method for imaging membrane potential. The voltage dependence of SHG by four different probes, three retinoids (all-trans retinal), and two new retinal analogs, 3-methyl-7-(4'-dimethylamino-phenyl)-2,4,6-heptatrienal (AR-3) and 3,7-dimethyl-9-(4'-dimethylamino-phenyl)-2,4,6,8-nonatetraenal (AR-4), and a styryl dye (FM4-64), were compared in HEK-293 cells. Results were analyzed by fitting data with an expression based on an electrooptic mechanism for SHG, which depends on the complex-valued first- and second-order nonlinear electric susceptibilities (χ² and χ³) of the probe. This gave values for the voltage sensitivity at the cell's resting potential, the voltage where the SHG is minimal, and the amplitude of the signal at that voltage for each of the four compounds. These measures show that χ² and χ³ are complex numbers for all compounds except all-trans retinal, consistent with the proximities of excitation and/or emission wavelengths to molecular resonances. Estimates of probe orientation and location in the membrane electric field show that, for the far-from-resonance case, the shot noise-limited signal/noise ratio depends on the location of the probe in the membrane, and on χ³ but not on χ².
authors.affiliation: University of Washington, Department of Physiology & Biophysics, Seattle, Washington, USA.
authors.forename: Patrick
authors.initials: P
authors.lastname: Theer
authors.forename: Winfried
authors.initials: W
authors.lastname: Denk
authors.forename: Mordechai
authors.initials: M
authors.lastname: Sheves
authors.forename: Aaron
authors.initials: A
authors.lastname: Lewis
authors.forename: Peter B
authors.initials: PB
authors.lastname: Detwiler
mesh_terms: D004560:Electricity; D057809:HEK293 Cells; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000379:methods*; D008564:Membrane Potentials / Q000502:physiology*; D011726:Pyridinium Compounds / Q000378:metabolism; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D012172:Retinaldehyde / Q000031:analogs & derivatives* / Q000378:metabolism; D013997:Time Factors
chemical_list: C092350:FM 4-64; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D012172:Retinaldehyde