Second harmonic imaging of membrane potential of neurons with retinal.

We present a method to optically measure and image the membrane potential of neurons, using the nonlinear optical phenomenon of second harmonic generation (SHG) with a photopigment retinal as the chromophore [second harmonic retinal imaging of membrane potential (SHRIMP)]. We show that all-trans retinal, when adsorbed to the plasma membrane of living cells, can report on the local electric field via its change in SHG. Using a scanning mode-locked Ti-sapphire laser, we collect simultaneous two-photon excited fluorescence (TPEF) and SHG images of retinal-stained kidney cells and cultured pyramidal neurons. Patch clamp experiments on neurons stained with retinal show an increase of 25% in SHG intensity per 100-mV depolarization. Our data are the first demonstration of optical measurements of membrane potential of mammalian neurons with SHG. SHRIMP could have wide applicability in neuroscience and, by modifying rhodopsin, could in principle be subject for developing genetically engineered voltage sensors.

[1]  Daniel A Dombeck,et al.  Optical Recording of Action Potentials with Second-Harmonic Generation Microscopy , 2004, The Journal of Neuroscience.

[2]  W. Webb,et al.  Optical imaging of cell membrane potential changes induced by applied electric fields. , 1986, Biophysical journal.

[3]  G. Wald.  The Molecular Basis of Visual Excitation , 1968, Nature.

[4]  W. Webb,et al.  Mechanism of the membrane potential sensitivity of the fluorescent membrane probe merocyanine 540. , 1978, Biochemistry.

[5]  M. Sheves,et al.  A comparison of the second harmonic generation from light-adapted, dark-adapted, blue, and acid purple membrane. , 1994, Biophysical journal.

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

[7]  Jerome Mertz,et al.  Mechanisms of membrane potential sensing with second-harmonic generation microscopy. , 2003, Journal of biomedical optics.

[8]  W. N. Ross,et al.  Changes in absorption, fluorescence, dichroism, and birefringence in stained giant axons: Optical measurement of membrane potential , 1977, The Journal of Membrane Biology.

[9]  A. Lewis,et al.  Determination of the absolute orientation of the retinylidene chromophore in purple membrane by a second-harmonic interference technique. , 1989, Biophysical journal.

[10]  Shen,et al.  In situ determination of induced dipole moments of pure and membrane-bound retinal chromophores. , 1989, Physical review. A, General physics.

[11]  L. Loew,et al.  Fluorescent indicators of membrane potential: microspectrofluorometry and imaging. , 1989, Methods in cell biology.

[12]  W. Denk,et al.  Two-photon laser scanning fluorescence microscopy. , 1990, Science.

[13]  L. Stryer,et al.  Retinal has a highly dipolar vertically excited singlet state: implications for vision. , 1976, Proceedings of the National Academy of Sciences of the United States of America.

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

[15]  L Cohen,et al.  Special Topic: Optical Approaches to Neuron Function , 1989 .

[16]  Jerome Mertz,et al.  Electro-optic response of second-harmonic generation membrane potential sensors , 2003 .

[17]  Theo Rasing,et al.  Second harmonic generation from Langmuir-Blodgett films of retinal and retinal Schiff bases , 1988 .

[18]  L M Loew,et al.  Second-harmonic imaging microscopy of living cells. , 2001, Journal of biomedical optics.

[19]  W. Webb,et al.  Multiphoton microscopy in biological research. , 2001, Current opinion in chemical biology.

[20]  W. N. Ross,et al.  Simultaneous optical measurements of electrical activity from multiple sites on processes of cultured neurons. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[21]  W. Webb,et al.  Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[22]  Watt W. Webb,et al.  Uniform polarity microtubule assemblies imaged in native brain tissue by second-harmonic generation microscopy , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[23]  G. Banker,et al.  Culturing nerve cells , 1998 .

[24]  Leslie M. Loew,et al.  Second-harmonic generation of biological interfaces: probing the membrane protein bacteriorhodopsin and imaging membrane potential around GFP molecules at specific sites in neuronal cells of C. elegans , 1999 .

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

[26]  K. Nakanishi,et al.  Movement of retinal along the visual transduction path. , 2000, Science.

[27]  B. Zemelman,et al.  Selective Photostimulation of Genetically ChARGed Neurons , 2002, Neuron.

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

[29]  Rafael Yuste,et al.  Imaging neurons : a laboratory manual , 1999 .

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

[31]  J Mertz,et al.  Membrane imaging by simultaneous second-harmonic generation and two-photon microscopy. , 2000, Optics letters.

[32]  J. Oudar,et al.  Hyperpolarizabilities of the nitroanilines and their relations to the excited state dipole moment , 1977 .

[33]  D. Schmid,et al.  Novel hyperpolarizable and fluorescent dyes in lipid membranes: studying membrane potentials using nonlinear optical and fluorescence , 2003 .

[34]  H Takei,et al.  Bacteriorhodopsin oriented in polyvinyl alcohol films as an erasable optical storage medium. , 1991, Applied optics.

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

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

[37]  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.

[38]  R. Frostig,et al.  Optical imaging of neuronal activity. , 1988, Physiological reviews.

[39]  Jerome Mertz,et al.  Photoinduced flip-flop of amphiphilic molecules in lipid bilayer membranes. , 2002, Physical review letters.

[40]  Rafael Yuste,et al.  A two-photon and second-harmonic microscope. , 2003, Methods.

[41]  R. Birge.  Photophysics and molecular electronic applications of the rhodopsins. , 1990, Annual review of physical chemistry.

[42]  Leslie M Loew,et al.  Second harmonic imaging microscopy. , 2003, Methods in enzymology.

[43]  R. Birge,et al.  Photophysics of light transduction in rhodopsin and bacteriorhodopsin. , 1981, Annual review of biophysics and bioengineering.

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

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

[46]  Leslie M Loew,et al.  Direct measurement of the voltage sensitivity of second-harmonic generation from a membrane dye in patch-clamped cells. , 2003, Optics letters.

[47]  G. Rayfield,et al.  Hyper-Rayleigh light scattering from an aqueous suspension of purple membrane. , 1994, Applied optics.

[48]  L. Loew,et al.  Second Harmonic Imaging Microscopy , 2003, Microscopy and Microanalysis.