Second Harmonic Generation in Neurons: Electro-Optic Mechanism of Membrane Potential Sensitivity
Jiang Jiang 1, Kenneth B Eisenthal 1 and Rafael Yuste 1*
1 Columbia University
* To whom correspondence should be addressed. E-mail: rmy5{at}columbia.edu.
Submitted on April 18, 2007
Revised on May 28, 2007
Accepted on 21 June 2007
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Abstract |
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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 ~36 °tilt angle of the chromophore to the membrane normal. Changes in membrane potential do not affect the polarization of the SHG signal. Finally, the voltage response of FM 4-64 is faster than 1 msec and does not reverse sign when imaged at either side of its absoption peak. We conclude that FM 4-64 senses membrane potential through an electro-optic mechanism, without significant chromophore membrane reorientation, redistribution, or spectral shift.
Key Words:
FM-464, SHG, membrane, neurons, optical, voltage
