Direct measurement of the voltage sensitivity of second-harmonic generation from a membrane dye in patch-clamped cells.

We report what is to our knowledge the first optical imaging of voltage-clamped cells by second-harmonic generation. For the membrane-staining styryl dye di-4-ANEPPS, we determined the sensitivity of second-harmonic generation to be 18%/100 mV at an excitation wavelength of 850 ns. This sensitivity is significantly better than the optimal 10%/100 mV under fluorescence and further establishes the importance of second-harmonic generation for the functional imaging of membrane potential in living cells.

[1]  M Deutsch,et al.  Connective tissue polarity. Optical second-harmonic microscopy, crossed-beam summation, and small-angle scattering in rat-tail tendon. , 1986, Biophysical journal.

[2]  L M Loew,et al.  High-resolution nonlinear optical imaging of live cells by second harmonic generation. , 1999, Biophysical journal.

[3]  Geraldine L. Richmond,et al.  Examination of the Electrochemical Interface between Two Immiscible Electrolyte Solutions by Second Harmonic Generation , 1997 .

[4]  L M Loew,et al.  Probing membrane potential with nonlinear optics. , 1993, Biophysical journal.

[5]  Bruce J Tromberg,et al.  Selective corneal imaging using combined second-harmonic generation and two-photon excited fluorescence. , 2002, Optics letters.

[6]  M Linial,et al.  Nonlinear optical measurement of membrane potential around single molecules at selected cellular sites. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[7]  L. Loew,et al.  Nonlinear optical properties of potential sensitive styryl dyes. , 1988, Biophysical journal.

[8]  L M Loew,et al.  Membrane electric properties by combined patch clamp and fluorescence ratio imaging in single neurons. , 1998, Biophysical journal.

[9]  L. Loew,et al.  Infrared nonlinear optical measurements of membrane potential in photoreceptor cells. , 1996, Biophysical journal.

[10]  Y. Shen,et al.  Surface properties probed by second-harmonic and sum-frequency generation , 1989, Nature.

[11]  S. Scully,et al.  Evidence for a charge-shift electrochromic mechanism in a probe of membrane potential , 1979, Nature.

[12]  William A Mohler,et al.  Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues. , 2002, Biophysical journal.

[13]  Satoshi Kawata,et al.  Second-harmonic-generation microscope with a microlens array scanner. , 2002, Optics letters.

[14]  Yan Liu,et al.  New Method for Determination of Surface Potential of Microscopic Particles by Second Harmonic Generation , 1998 .

[15]  L M Loew,et al.  Design and characterization of electrochromic membrane probes. , 1982, Journal of biochemical and biophysical methods.

[16]  K. Eisenthal,et al.  Liquid Interfaces Probed by Second-Harmonic and Sum-Frequency Spectroscopy. , 1996, Chemical reviews.

[17]  Jung Y. Huang,et al.  Second-harmonic generation in purple membrane−poly(vinyl alcohol) films: probing the dipolar characteristics of the bacteriorhodopsin chromophore in bR570 and M412 , 1989 .

[18]  L M Loew,et al.  Spectra, membrane binding, and potentiometric responses of new charge shift probes. , 1985, Biochemistry.

[19]  J Mertz,et al.  Coherent scattering in multi-harmonic light microscopy. , 2001, Biophysical journal.

[20]  G. Salama,et al.  A naphthyl analog of the aminostyryl pyridinium class of potentiometric membrane dyes shows consistent sensitivity in a variety of tissue, cell, and model membrane preparations , 1992, The Journal of Membrane Biology.

[21]  Jerome Mertz,et al.  Membrane imaging by second-harmonic generation microscopy , 2000 .

[22]  Maria Goeppert-Mayer.  Über Elementarakte mit zwei Quantensprüngen , 1931 .

[23]  F Bezanilla,et al.  Charge-shift probes of membrane potential. Characterization of aminostyrylpyridinium dyes on the squid giant axon. , 1985, Biophysical journal.