This Article Full Text Full Text (PDF) Supplement All Versions of this Article: biophysj.107.111021v1 93/5/L26    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Jiang, J. Articles by Yuste, R. Search for Related Content PubMed PubMed Citation Articles by Jiang, J. Articles by Yuste, R.

Second Harmonic Generation in Neurons: Electro-Optic Mechanism of Membrane Potential Sensitivity

Jiang Jiang ^*, Kenneth B. Eisenthal  and Rafael Yuste ^*

^* Howard Hughes Medical Institute, Department of Biological Sciences, and Department of Chemistry, Columbia University, New York

Correspondence: Address reprint requests and inquiries to R. Juste or K. B. Eisenthal, E-mail: rmy5{at}columbia.edu; kbe1{at}columbia.edu.

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. 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.