Second−harmonic generation imaging of membrane potential with retinal analogues

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−(40−dimethylamino−phenyl)− 2,4,6−heptatrienal (AR−3) and 3,7−dimethyl−9−(40−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 (c2 and c3) 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 c2 and c3 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−fromresonance case, the shot noise−limited signal/noise ratio depends on the location of the probe in the membrane, and on c3 but not on c