title: Second harmonic generation in neurons: electro-optic mechanism of membrane potential sensitivity.
abstract: Second harmonic generation (SHG) from membrane-bound chromophores can be used to image membrane potential in neurons. We investigate the biophysical mechanism responsible for the SHG voltage sensitivity of the styryl dye FM 4-64 in pyramidal neurons from mouse neocortical slices. SHG signals are exquisitely sensitive to the polarization of the incident laser light. Using this polarization sensitivity in two complementary approaches, we estimate a approximately 36 degrees tilt angle of the chromophore to the membrane normal. Changes in membrane potential do not affect the polarization of the SHG signal. The voltage response of FM 4-64 is faster than 1 ms and does not reverse sign when imaged at either side of its absorption peak. We conclude that FM 4-64 senses membrane potential through an electro-optic mechanism, without significant chromophore membrane reorientation, redistribution, or spectral shift.
authors.affiliation: Howard Hughes Medical Institute, Department of Biological Sciences, Columbia University, New York, NY, USA.
authors.forename: Jiang
authors.initials: J
authors.lastname: Jiang
authors.forename: Kenneth B
authors.initials: KB
authors.lastname: Eisenthal
authors.forename: Rafael
authors.initials: R
authors.lastname: Yuste
mesh_terms: D000200:Action Potentials; D000818:Animals; D001703:Biophysics / Q000379:methods*; D002138:Calibration; D004563:Electrochemistry / Q000379:methods; D004566:Electrodes; D007700:Kinetics; D007834:Lasers; D008564:Membrane Potentials; D008954:Models, Biological; D015233:Models, Statistical; D009474:Neurons / Q000378:metabolism*; D055095:Optics and Photonics