Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms


Although the nonlinear optical effect known as second-harmonic generation (SHG) has been recognized since the earliest days of laser physics and was demonstrated through a microscope over 25 years ago, only in the past few years has it begun to emerge as a viable microscope imaging contrast mechanism for visualization of cell and tissue structure and function. Only small modifications are required to equip a standard laser-scanning two-photon microscope for second-harmonic imaging microscopy (SHIM). Recent studies of the three-dimensional in vivo structures of well-ordered protein assemblies, such as collagen, microtubules and muscle myosin, are beginning to establish SHIM as a nondestructive imaging modality that holds promise for both basic research and clinical pathology. Thus far the best signals have been obtained in a transmitted light geometry that precludes in vivo measurements on large living animals. This drawback may be addressed through improvements in the collection of SHG signals via an epi-illumination microscope configuration. In addition, SHG signals from certain membrane-bound dyes have been shown to be highly sensitive to membrane potential. Although this indicates that SHIM may become a valuable tool for probing cell physiology, the small signal size would limit the number of photons that could be collected during the course of a fast action potential. Better dyes and optimized microscope optics could ultimately lead to the imaging of neuronal electrical activity with SHIM.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type



Prices may be subject to local taxes which are calculated during checkout

Figure 1: Endogenous SHG images of native mouse leg.
Figure 2: Endogenous SHG imaging of a living adult C. elegans nematode, showing two distinct axial slices.
Figure 3: Membrane potential sensitivity of SHG from di-4-ANEPPS on a voltage-clamped N1E-115 neuroblastoma cell.


  1. Freund, I., Deutsch, M. & Sprecher, A. Connective tissue polarity. Optical second harmonic microscopy, crossed-beam summation, and small-angle scattering in rat tail tendon. Biophys. J. 50, 693–712 (1986).

    Article  CAS  Google Scholar 

  2. Huang, Y.A., Lewis, A. & Loew, L.M. Nonlinear optical properties of potential sensitive styryl dyes. Biophys. J. 53, 665–670 (1988).

    Article  CAS  Google Scholar 

  3. Rasing, T., Huang, J., Lewis, A., Stehlin, T. & Shen, Y.R. In situ determination of induced dipole moments of pure and membrane-bound retinal chromophores. Phys. Rev. A 40, 1684–1687 (1989).

    Article  CAS  Google Scholar 

  4. Campagnola, P.J., Wei, M.–d., Lewis, A. & Loew, L.M. High-resolution optical imaging of live cells by second harmonic generation. Biophys. J. 77, 3341–3349 (1999).

    Article  CAS  Google Scholar 

  5. Moreaux, L., Sandre, O., Blanchard–Desce, M. & Mertz, J. Membrane imaging by simultaneous second harmonic generation and two-photo microscopy. Optics Lett. 25, 320–322 (2000).

    Article  CAS  Google Scholar 

  6. Campagnola, P.J. et al. Three-dimensional high-resolution second harmonic generation imaging of endogenous structural proteins in biological tissues. Biophys. J. 82, 493–508 (2002).

    Article  CAS  Google Scholar 

  7. Zoumi, A., Yeh, A. & Tromberg, B.J. Imaging cells and extracellular matrix in vivo by using second harmonic generation and two-photon excited fluorescence. Proc. Natl. Acad. Sci. USA 99, 11014–11019 (2002).

    Article  CAS  Google Scholar 

  8. Yeh, A.T., Nassif, N., Zoumi, A. & Tromberg, B.J. Selective corneal imaging using combined second harmonic generation and two-photon excited fluorescence. Optics Lett. 27, 2082–2084 (2002).

    Article  CAS  Google Scholar 

  9. Zipfel, W.R. et al. Live tissue intrinsic emission microscopy using multiphoton–excited native fluorescence and second harmonic generation. Proc. Natl. Acad. Sci. USA 100, 7075–7080 (2003).

    Article  CAS  Google Scholar 

  10. Dombeck, D.A. et al. Uniform polarity microtubule assemblies imaged in native brain tissue by second harmonic generation microscopy. Proc. Natl. Acad. Sci. USA 100, 7081–7086 (2003).

    Article  CAS  Google Scholar 

  11. Brown, E. et al. Dynamic imaging of collagen and its modulation in tumors in vivo using second harmonic generation. Nat. Med. 9, 796–800 (2003).

    Article  CAS  Google Scholar 

  12. Hellwarth, R. & Christensen, P. Nonlinear optical microscopic examination of structure in polycrystalline ZnSe. Optics Comm. 12, 318–322 (1974).

    Article  CAS  Google Scholar 

  13. Sheppard, C.J.R., Kompfner, R., Gannaway, J. & Walsh, D. Scanning harmonic optical microscope. IEEE J. Quantum Electron. 13E, 100D (1977).

    Google Scholar 

  14. Bouevitch, O., Lewis, A., Pinevsky, I., Wuskell, J.P. & Loew, L.M. Probing membrane potential with non-linear optics. Biophys. J. 65, 672–679 (1993).

    Article  CAS  Google Scholar 

  15. Ben-Oren, I., Peleg, G., Lewis, A., Minke, B. & Loew, L.M. Infrared nonlinear optical measurements of membrane potential in photoreceptor cells. Biophys. J. 71, 1616–1620 (1996).

    Article  CAS  Google Scholar 

  16. Moreaux, L., Sandre, O. & Mertz, J. Membrane imaging by second harmonic generation microscopy. J. Opt. Soc. Am. B 17, 1685–1694 (2000).

    Article  CAS  Google Scholar 

  17. Mohler, W., Millard, A.C. & Campagnola, P.J. Second harmonic generation imaging of endogenous structural proteins. Methods 29, 97–109 (2003).

    Article  CAS  Google Scholar 

  18. Stoller, P., Reiser, K.M., Celliers, P.M. & Rubenchik, A.M. Polarization-modulated second harmonic generation in collagen. Biophys. J. 82, 3330–3342 (2002).

    Article  CAS  Google Scholar 

  19. Stoller, P., Kim, B.M., Rubenchik, A.M., Reiser, K.M. & Da Silva, L.B. Polarization-dependent optical second harmonic imaging of a rat-tail tendon. J. Biomed. Opt. 7, 205–214 (2002).

    Article  Google Scholar 

  20. Campagnola, P.J., Clark, H.A., Mohler, W.A., Lewis, A. & Loew, L.M. Second harmonic imaging microscopy of living cells. J. Biomed. Opt. 6, 277–286 (2001).

    Article  CAS  Google Scholar 

  21. Millard, A.C., Campagnola, P.J., Mohler, W., Lewis, A. & Loew, L.M. Second harmonic imaging microscopy. Methods Enzymol. 361, 47–69 (2003).

    Article  CAS  Google Scholar 

  22. Cox, G. et al. 3-dimensional imaging of collagen using second harmonic generation. J. Struct. Biol. 141, 53–62 (2003).

    Article  CAS  Google Scholar 

  23. Inoue, S. Video Microscopy (Plenum Press, New York, 1986).

    Book  Google Scholar 

  24. Brown, E. et al. Dynamic imaging of collagen and its modulation in tumors in vivo using second harmonic generation. Nat. Med. 9, 796–800 (2003).

    Article  CAS  Google Scholar 

  25. Loew, L.M. in Fluorescent and Luminescent Probes for Biological Activity (ed. Mason, W.T.) 210–221 (Academic Press, London, 1999).

    Book  Google Scholar 

  26. Huang, J.Y., Lewis, A. & Loew, L.M. Non-linear optical properties of potential sensitive styryl dyes. Biophys. J. 53, 665–670 (1988).

    Article  CAS  Google Scholar 

  27. Clark, H.A., Campagnola, P.J., Wuskell, J.P., Lewis, A. & Loew, L.M. Second harmonic generation properties of fluorescent polymer encapsulated gold nanoparticles. J. Am. Chem. Soc. 122, 10234–10235 (2000).

    Article  CAS  Google Scholar 

  28. Moreaux, L., Sandre, O., Charpak, S., Blanchard–Desce, M. & Mertz, J. Coherent scattering in multi-harmonic light microscopy. Biophys. J. 80, 1568–1574 (2001).

    Article  CAS  Google Scholar 

  29. Millard, A.C., Jin, L., Lewis, A. & Loew, L.M. Direct measurement of the voltage sensitivity of second harmonic generation from a membrane dye in patch-clamped cells. Optics Lett. 28, 1221–1223 (2003).

    Article  CAS  Google Scholar 

  30. Moreaux, L., Pons, T., Dambrin, V., Blanchard–Desce, M. & Mertz, J. Electro–optic response of second harmonic generation membrane potential sensors. Optics Lett. 28, 625–627 (2003).

    Article  CAS  Google Scholar 

  31. Peleg, G. et al. Gigantic optical non-linearities from nanoparticle enhanced molecular probes with potential for selectively imaging the structure and physiology of nanometric regions in cellular systems. Bioimaging 4, 215–224 (1996).

    Article  Google Scholar 

  32. Peleg, G., Lewis, A., Linial, M. & Loew, L.M. Nonlinear optical measurement of membrane potential around single molecules at selected cellular sites. Proc. Natl. Acad. Sci. USA 96, 6700–6704 (1999).

    Article  CAS  Google Scholar 

Download references


The authors would like to thank their collaborators Aaron Lewis, Andrew Millard, William Mohler, Heather Clark and David Boudreau for their contributions to this work. We gratefully acknowledge financial support under US Office of Naval Research grant no. N0014-98-1-0703, National Institute of Biomedical Imaging and Bioengineering grant no. R01EB00196 and National Center for Research Resources grant no. R21 RR13472.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Leslie M Loew.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Campagnola, P., Loew, L. Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms. Nat Biotechnol 21, 1356–1360 (2003).

Download citation

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing