Second Harmonic Imaging of Membrane Potential

  • Leslie M. Loew
  • Aaron Lewis


The non-linear optical effect known as second harmonic generation (SHG) has been recognized since the earliest days of the laser. But it has only been in the last few years that it has begun to emerge as a viable microscope imaging contrast mechanism for visualization of cell and tissue structure and function. This is because only small modifications are required to equip a standard laser scanning 2-photon microscope for second harmonic imaging microscopy (SHIM). SHG signals from certain membrane-bound dyes are highly sensitive to membrane potential, indicating that SHIM may become a valuable probe of cell physiology. However, for the current generation of dyes and microscopes, the small signal size limits the number of photons that can be collected during the course of a fast action potential. Better dyes and optimized microscope optics could ultimately lead to the ability to image neuronal electrical activity with SHIM.


Second Harmonic Generation Second Harmonic Generation Signal Styryl Quinolinium Donor Moiety Second Harmonic Generation Intensity 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



We are indebted to the many talented students and colleagues who have participated in the development of SHG imaging in our laboratories over the last 20 years. This work was supported by NIH EB001963.


  1. Araya R, Jiang J, Eisenthal KB, Yuste R (2006) The spine neck filters membrane potentials. Proc Natl Acad Sci USA 103:17961–17966.PubMedCrossRefGoogle Scholar
  2. Araya R, Nikolenko V, Eisenthal KB, Yuste R (2007) Sodium channels amplify spine potentials. Proc Natl Acad Sci USA 104:12347–12352.PubMedCrossRefGoogle Scholar
  3. Ben-Oren I, Peleg G, Lewis A, Minke B, Loew LM (1996) Infrared nonlinear optical measurements of membrane potential in photoreceptor cells. Biophys J 71:1616–1620.PubMedCrossRefGoogle Scholar
  4. Bianchini P, Diaspro A (2008) Three-dimensional (3D) backward and forward second harmonic generation (SHG) microscopy of biological tissues. J Biophotonics 1:443–450.PubMedCrossRefGoogle Scholar
  5. Bouevitch O, Lewis A, Pinevsky I, Wuskell JP, Loew LM (1993) Probing membrane potential with non-linear optics. Biophys J 65:672–679.PubMedCrossRefGoogle Scholar
  6. Campagnola P, Loew LM (2003) Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms. Nat Biotechnol 21:1356–1360.PubMedCrossRefGoogle Scholar
  7. Campagnola PJ, Wei MD, Lewis A, Loew LM (1999) High resolution optical imaging of live cells by second harmonic generation. Biophys J 77:3341–3349.PubMedCrossRefGoogle Scholar
  8. Campagnola PJ, Clark HA, Mohler WA, Lewis A, Loew LM (2001) Second harmonic imaging microscopy of living cells. J Biomed Opt 6:277–286.PubMedCrossRefGoogle Scholar
  9. Clark HA, Campagnola PJ, Wuskell JP, Lewis A, Loew LM (2000) Second harmonic generation properties of fluorescent polymer encapsulated gold nanoparticles. J Am Chem Soc 122:10234–10235.CrossRefGoogle Scholar
  10. Cole JM, Kreiling S (2002) Exploiting structure/property relationships in organic non-linear optical materials: developing strategies to realize the potential of TCNQ derivatives. CrystEngComm 4:232–238.CrossRefGoogle Scholar
  11. Conboy JC, Richmond GL (1997) Examination of the electrochemical interface between two immiscible electrolye solutions by second harmonic generation. J Phys Chem B 101:983–990.CrossRefGoogle Scholar
  12. Denk W, Strickler JH, Webb WW (1990) Two-photon laser scanning fluorescence microscopy. Science 248:73–76.PubMedCrossRefGoogle Scholar
  13. Dirk CW, Twieg RJ, Wagniere J (1986) The contribution of Pi electrons to second harmonic generation in organic molecule. J Am Chem Soc 108:5387–5395.CrossRefGoogle Scholar
  14. Dombeck DA, Blanchard-Desce M, Webb WW (2004) Optical recording of action potentials with second-harmonic generation microscopy. J Neurosci 24:999–1003.PubMedCrossRefGoogle Scholar
  15. Dombeck DA, Sacconi L, Blanchard-Desce M, Webb WW (2005) Optical recording of fast neuronal membrane potential transients in acute mammalian brain slices by second-harmonic generation microscopy. J Neurophysiol 94:3628–3636.PubMedCrossRefGoogle Scholar
  16. Fluhler E, Burnham VG, Loew LM (1985) Spectra, membrane binding, and potentiometric responses of new charge shift probes. Biochemistry 24:5749–5755.PubMedCrossRefGoogle Scholar
  17. Freund I, Deutsch M, Sprecher A (1986) Connective tissue polarity. Optical second-harmonic microscopy, crossed-beam summation, and small-angle scattering in rat-tail tendon. Biophys J 50:693–712.PubMedCrossRefGoogle Scholar
  18. Huang JY, Lewis A, Loew LM (1988) Non-linear optical properties of potential sensitive styryl dyes. Biophys J 53:665–670.PubMedCrossRefGoogle Scholar
  19. Jiang J, Eisenthal KB, Yuste R (2007) Second harmonic generation in neurons: electro-optic mechanism of membrane potential sensitivity. Biophys J 93: L26–L28.PubMedCrossRefGoogle Scholar
  20. Millard AC, Campagnola PJ, Mohler W, Lewis A, Loew LM (2003a) Second harmonic imaging microscopy. In: Marriott G, Parker I (eds) Methods in enzymology, vol 361B. Academic Press, San Diego.Google Scholar
  21. Millard AC, Jin L, Lewis A, Loew LM (2003b) Direct measurement of the voltage sensitivity of second-harmonic generation from a membrane dye in patch-clamped cells. Opt Lett 28:1221–1223.PubMedCrossRefGoogle Scholar
  22. Millard AC, Jin L, et al (2004) Sensitivity of second harmonic generation from styryl dyes to trans-membrane potential. Biophys J 86:1169–1176.PubMedCrossRefGoogle Scholar
  23. Millard AC, Jin L, et al (2005a) Wavelength- and time-dependence of potentiometric non-linear optical signals from styryl dyes. J Memb Biol 208:103–111.CrossRefGoogle Scholar
  24. Millard AC, Lewis A, Loew LM (2005b) Second harmonic imaging of membrane potential. In Imaging in neuroscience and development. In: Yuste R, Lanni F, Konnerth A (eds) Imaging neurons a laboratory manual. Cold Spring Harbour Laboratory Press, New York.Google Scholar
  25. Mohler W, Millard AC, Campagnola PJ (2003) Second harmonic generation imaging of endogenous structural proteins. Methods 29:97–109.PubMedCrossRefGoogle Scholar
  26. Moreaux L, Sandre O, Blanchard-desce M, Mertz J (2000a) Membrane imaging by simultaneous second-harmonic generation and two-photo microscopy. Opt Lett 25:320–322.PubMedCrossRefGoogle Scholar
  27. Moreaux L, Sandre O, Mertz J (2000b) Membrane imaging by second harmonic generation microscopy. J Opt Soc Am B 17:1685–1694.CrossRefGoogle Scholar
  28. Moreaux L, Pons T, Dambrin V, Blanchard-Desce M, Mertz J (2003) Electro-optic response of second-harmonic generation membrane potential sensors. Opt Lett 28:625–627.PubMedCrossRefGoogle Scholar
  29. Morley JO (1988) Non-linear optical properties of organic molecules. 7. Calculated hyperpolarizabilities of azulenes and sesquifulvalene. J Am Chem Soc 110:7660–7663.CrossRefGoogle Scholar
  30. Nicoud JF, Twieg RJ (1987) Design and synthesis of organic molecular compounds for efficient second-harmonic generation. In Chemla DS, Zyss J (eds) Nonlinear optical properties of organic molecules and crystals. Academic Press Inc, Orlando.Google Scholar
  31. Nuriya M, Jiang J, Nemet B, Eisenthal KB, Yuste R (2006) Imaging membrane potential in dendritic spines. Proc Natl Acad Sci USA 103:786–790.PubMedCrossRefGoogle Scholar
  32. Pons T, Moreaux L, Mongin O, Blanchard-Desce M, Mertz J (2003) Mechanisms of membrane potential sensing with second-harmonic generation microscopy. J Biomed Opt 8:428–431.PubMedCrossRefGoogle Scholar
  33. Sacconi L, Dombeck DA, Webb WW (2006) Overcoming photodamage in second-harmonic generation microscopy: real-time optical recording of neuronal action potentials. Proc Natl Acad Sci U S A 103:3124–3129.PubMedCrossRefGoogle Scholar
  34. Sheppard C, Kompfner R, Gannaway J, Walsh D (1977). The scanning harmonic optical microscope. IEEE J Quan Electron 13:100D.Google Scholar
  35. Teisseyre TZ, Millard AC et al (2007) Nonlinear optical potentiometric dyes optimized for imaging with 1064-nm light. J Biomed Opt 12:044001.PubMedCrossRefGoogle Scholar
  36. Williams DJ (1984) Organic polymeric and non-polymeric materials with large optical non-linearities. Angew Chem Int Ed Engl 23:690–703.CrossRefGoogle Scholar
  37. Yan ECY, Liu Y, Eisenthal KB (1998) New method for determination of surface potential of microscopic particles by second harmonic generation. J Phys Chem B 102:6331–6336.CrossRefGoogle Scholar
  38. Yan P, Millard AC, Wei M, Loew LM (2006) Unique contrast patterns from resonance-enhanced chiral SHG of cell membranes. J Am Chem Soc 128:11030–11031.PubMedCrossRefGoogle Scholar
  39. Yan P, Xie A, Wei MD, Loew LM (2008) Amino(oligo)thiophene-based environmentally sensitive biomembrane chromophores. J Org Chem 73:6587–6594.PubMedCrossRefGoogle Scholar
  40. Zhou W-L, Yan P, Wuskell JP, Loew LM, Antic SD (2007) Intracellular long-wavelength voltage-sensitive dyes for studying the dynamics of action potentials in axons and thin dendrites. J Neurosci Meth 164:225–239.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Leslie M. Loew
    • 1
  • Aaron Lewis
    • 2
  1. 1.Department of Cell Biology, R. D. Berlin Center for Cell Analysis and ModelingUniversity of Connecticut Health CenterFarmingtonUSA
  2. 2.Department of Applied PhysicsHebrew UniversityJerusalemIsrael

Personalised recommendations