Second Harmonic OCT and Combined MPM/OCT

  • Zhongping ChenEmail author
  • Shuo Tang
Reference work entry


This chapter describes combined multiphoton microscopy (MPM) and optical coherence tomography (OCT) system and second harmonic OCT (SH-OCT) system. Second harmonic generation (SHG) enables direct imaging of optically anisotropic biological structures, such as membranes, structural proteins, microtubule ensembles, and collagen. SH-OCT combines molecular contrast of SHG with coherence gating of OCT. MPM/OCT combines molecular contrast of MPM with scattering contrast of OCT. Combining MPM and OCT onto a single platform creates a novel multimodality image technique which can acquire structural and functional imaging of tissues simultaneously. We will review the principle and technology of SH-OCT and combined MPM/OCT system, and illustrate a few examples of its applications.


MPM Multimodal imaging Multiphoton microscopy Second harmonic Second harnomic OCT SH-OCT 



We would like to thank many of our colleagues who have contributed to the MPM/OCT and SH-OCT projects at UCI and UBC. We want to acknowledge grant support from the National Institutes of Health (R01EB-00293, R01CA-91717, R01EB-10090, R01EY-021519, R01HL-105215, P41EB-015890), Air Force Office of Scientific Research (F49620-00-1-0371), the Beckman Laser Institute Endowment, Natural Sciences and Engineering Research Council of Canada, and the Canada Foundation for Innovation.


  1. 1.
    D. Huang, E.A. Swanson, C.P. Lin, J.S. Schuman, W.G. Stinson, W. Chang, M.R. Hee, T. Flotte, K. Gregory, C.A. Puliafito, J.G. Fujimoto, Optical coherence tomography. Science 254(5035), 1178–1181 (1991)CrossRefADSGoogle Scholar
  2. 2.
    M.R. Lee, J.A. Izatt, E.A. Swanson, D. Huang, J.S. Schumun, C.P. Lin, C.A. Puliafito, J.G. Fujimoto, Optical coherence tomography for ophthalmic imaging: new technique delivers micron-scale resolution. IEEE Eng. Med. Biol. Mag. 14(1), 67–76 (1995)CrossRefGoogle Scholar
  3. 3.
    W. Denk, J.H. Strickler, W.W. Webb, Two-photon laser scanning fluorescence microscopy. Science 248, 73–79 (1990)CrossRefADSGoogle Scholar
  4. 4.
    W.R. Zipfel, R.M. Williams, W.W. Webb, Nonlinear magic: multiphoton microscopy in the biosciences. Nat. Biotechnol. 21, 1369–1377 (2003)CrossRefGoogle Scholar
  5. 5.
    W.R. Zipfel, R.M. Williams, R.A. Christie, Y. Nikitin, B.T. Hyman, Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation. Proc. Natl. Acad. Sci. USA 100, 7075 (2003)CrossRefADSGoogle Scholar
  6. 6.
    C.K. Sun, C.C. Chen, S.W. Chu, T.H. Tsai, Y.C. Chen, B.L. Lin, Multiharmonic-generation biopsy of skin. Opt. Lett. 28, 2488 (2003)CrossRefADSGoogle Scholar
  7. 7.
    D. Kobat, N.G. Horton, C. Xu, In vivo two-photon microscopy to 1.6-mm depth in mouse cortex. J. Biomed. Opt. 16(10), 106014 (2011)CrossRefADSGoogle Scholar
  8. 8.
    E. Beaurepaire, L. Moreaux, F. Amblard, J. Mertz, Combined scanning optical coherence and two-photon-excited fluorescence microscopy. Opt. Lett. 24, 969–971 (1999)CrossRefADSGoogle Scholar
  9. 9.
    S. Tang, T.B. Krasieva, Z. Chen, B. Tromberg, Combined multiphoton microscopy and optical coherence tomography using a 12 femtosecond, broadband source. J. Biomed. Opt. 11, 020502 (2006)CrossRefADSGoogle Scholar
  10. 10.
    S. Tang, C.H. Sun, T.B. Krasieva, Z. Chen, B. Tromberg, Imaging sub-cellular scattering contrast using combined optical coherence and multiphoton microscopy. Opt. Lett. 32, 503–505 (2007)CrossRefADSGoogle Scholar
  11. 11.
    C. Vinegoni, T.S. Ralston, W. Tan, W. Luo, D.L. Marks, S.A. Boppart, Integrated structural and functional optical imaging combining spectral-domain optical coherence and multiphoton microscopy. Appl. Phys. Lett. 88, 053901 (2006)CrossRefADSGoogle Scholar
  12. 12.
    C. Joo, K.H. Kim, J.F. de Boer, Spectral-domain optical coherence phase and multiphoton microscopy. Opt. Lett. 32, 623–625 (2007)CrossRefADSGoogle Scholar
  13. 13.
    S. Yazdanfar, Y.Y. Chen, P.T.C. So, L.H. Laiho, Multifunctional imaging of endogenous contrast by simultaneous nonlinear and optical coherence microscopy of thick tissues. Micr. Res. Tech. 70, 503–505 (2007)CrossRefGoogle Scholar
  14. 14.
    Y. Jiang, I. Tomov, Y. Wang, Z. Chen, Second harmonic optical coherence tomography. Opt. Lett. 29, 1090–1092 (2004)CrossRefADSGoogle Scholar
  15. 15.
    Y. Jiang, I. Tomov, Y. Wang, Z. Chen, High-resolution second-harmonic optical coherence tomography of collagen in rat-tail tendon. Appl. Phys. Lett. 86, 133901 (2005)CrossRefADSGoogle Scholar
  16. 16.
    S. Yazdanfar, L.H. Laiho, P.T.C. So, Interferometric second harmonic generation microscopy. Opt. Express 12, 2739 (2004)CrossRefADSGoogle Scholar
  17. 17.
    B.E. Applegate, C. Yang, A.M. Rollins, J.A. Izatt, Polarization-resolved second-harmonic-generation optical coherence tomography in collagen. Opt. Lett. 29, 2252–2254 (2004)CrossRefADSGoogle Scholar
  18. 18.
    M.V. Sarunic, B.E. Applegate, J.A. Izatt, Spectral domain second harmonic optical coherence tomography. Opt. Lett. 30, 2391–2393 (2005)CrossRefADSGoogle Scholar
  19. 19.
    J. Su, I.V. Tomov, Y. Jiang, Z. Chen, High resolution frequency-domain second-harmonic optical coherence tomography. Appl. Opt. 46, 1770–1775 (2007)CrossRefADSGoogle Scholar
  20. 20.
    A. Zoumi, A. Yeh, B.J. Tromberg, Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence. Proc. Natl. Acad. Sci. USA 99, 11014 (2002)CrossRefADSGoogle Scholar
  21. 21.
    P.J. Campagnola, M.D. Wei, A. Lewis, L.M. Loew, High-resolution nonlinear optical imaging of live cells by second harmonic generation. Biophys. J. 77, 3341–3349 (1999)CrossRefGoogle Scholar
  22. 22.
    P.J. Campagnola, L.M. Loew, Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms. Nat. Biotechnol. 21, 1356–1360 (2003)CrossRefGoogle Scholar
  23. 23.
    Y.R. Shen, The Principles of Nonlinear Optics (Wiley, New York, 1984)Google Scholar
  24. 24.
    C. Xu, W.R. Zipfel, J.B. Shear, R.M. Williams, W.W. Webb, Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy. Proc. Natl. Acad. Sci. USA 93, 10763–10768 (1996)CrossRefADSGoogle Scholar
  25. 25.
    K. Konig, Multiphoton microscopy in life sciences. J. Microsc. 200, 83–104 (2000)CrossRefGoogle Scholar
  26. 26.
    G. Liu, Z. Chen, Fiber-based combined optical coherence and multiphoton endomicroscopy. J. Biomed. Opt. 16(3), 036010 (2011)CrossRefADSGoogle Scholar
  27. 27.
    B.W. Graf, S.A. Boppart, Multimodal in vivo skin imaging with integrated optical coherence and multiphoton microscopy. IEEE J. Sel. Top. Quantum Electron. 18, 1280–1286 (2012)CrossRefGoogle Scholar
  28. 28.
    M. Müller, J. Squier, G.J. Brakenhoff, Measurement of femtosecond pulses in the focal point of a high-numerical-aperture lens by two-photon absorption. Opt. Lett. 20, 1038–1040 (1995)CrossRefADSGoogle Scholar
  29. 29.
    M. Müller, J. Squier, R. Wolleschensky, U. Simon, G.J. Brakenhoff, Dispersion pre-compensation of 15 femtosecond optical pulses for high-numerical-aperture objectives. J. Microsc. 191, 141–150 (1998)CrossRefGoogle Scholar
  30. 30.
    A.F. Fercher, W. Drexler, C.K. Hizenberger, Optical coherence tomography–principles and applications. Rep. Prog. Phys. 66, 239–303 (2003)CrossRefADSGoogle Scholar
  31. 31.
    B. Jeong, B. Lee, M.S. Jang, H. Nam, S.J. Yoon, T. Wang, J. Doh, B.G. Yang, M.H. Jang, K.H. Kim, Combined two-photon microscopy and optical coherence tomography using individually optimized sources. Opt. Express 19(14), 13089–13096 (2011)CrossRefADSGoogle Scholar
  32. 32.
    S. Tang, Y. Zhou, K.K. Chan, T. Lai, Multiscale multimodal imaging with multiphoton microscopy and optical coherence tomography. Opt. Lett. 36(24), 4800–4802 (2011)CrossRefADSGoogle Scholar
  33. 33.
    S. Tang, Y. Zhou, M.J. Ju, Multimodal optical imaging with multiphoton microscopy and optical coherence tomography. J. Biophotonics 5(5–6), 396–403 (2012)CrossRefGoogle Scholar
  34. 34.
    M.T. Myaing, D.G. MacDonald, L. Xingde, Fiber-optic scanning two-photon fluorescence endoscope. Opt. Lett. 31, 1076–1078 (2006)CrossRefADSGoogle Scholar
  35. 35.
    L. Fu, A. Jain, H. Xie, C. Cranfield, M. Gu, Nonlinear optical endoscopy based on a double clad photonic crystal fiber and a MEMS mirror. Opt. Express 14, 1027–1032 (2006)CrossRefADSGoogle Scholar
  36. 36.
    W. Jung, S. Tang, D.T. McCormic, T. Xie, Y.C. Ahn, J. Su, I.V. Tomov, T.B. Krasieva, B.J. Tromberg, Z. Chen, Miniaturized probe based on a microelectromechanical system mirror for multiphoton microscopy. Opt. Lett. 33(12), 1324–1326 (2008)CrossRefADSGoogle Scholar
  37. 37.
    G. Liu, T. Xie, I.V. Tomov, J. Su, L. Yu, J. Zhang, B.J. Tromberg, Z. Chen, Rotational multiphoton endoscopy with a 1 micron fiber laser system. Opt. Lett. 34(15), 2249–2251 (2009)CrossRefADSGoogle Scholar
  38. 38.
    S. Tang, W. Jung, D. McCormick, T. Xie, J. Su, Y.C. Ahn, B.J. Tromberg, Z. Chen, Design and implementation of fiber-based multiphoton endoscopy with microelectromechanical systems scanning. J. Biomed. Opt. 14(3), 034005 (2009)CrossRefADSGoogle Scholar
  39. 39.
    G. Liu, K. Kieu, F.W. Wise, Z. Chen, Multiphoton microscopy system with a compact fiber-based femtosecond-pulse laser and handheld probe. J. Biophotonics 4(1–2), 34–39 (2011)CrossRefGoogle Scholar
  40. 40.
    I. Freund, M. Deutsch, Second-harmonic microscopy of biological tissue. Opt. Lett. 11, 94–96 (1986)CrossRefADSGoogle Scholar
  41. 41.
    J. Mertz, L. Moreausx, Second-harmonic generation by focused excitation of inhomogeneously distributed scatterers. Opt. Commun. 196, 325–330 (2001)CrossRefADSGoogle Scholar
  42. 42.
    L. Moreaux, O. Sandre, L. Mertz, Membrane imaging by second-harmonic generation microscopy. J. Opt. Soc. Am. B 17, 1685 (2000)CrossRefADSGoogle Scholar
  43. 43.
    P.J.A. Campagnola, C. Millard, M. Terasaki, P.E. Hoppe, C.J. Malone, W.A. Mohler, Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues. Biophys. J. 82, 493 (2002)CrossRefGoogle Scholar
  44. 44.
    D.A. Dombeck, K.A. Kasischke, H.D. Vishwasrao, M. Ingelsson, B.T. Hyman, W.W. Webb, Uniform polarity microtubule assemblies imaged in native brain tissue by second-harmonic generation microscopy. Proc. Natl. Acad. Sci. USA 100, 7081–7086 (2003)CrossRefADSGoogle Scholar
  45. 45.
    G. Cox, E. Kable, A. Jones, I.K. Fraser, F. Manconi, M.D. Gorrell, 3-dimensional imaging of collagen using second harmonic generation. J. Struct. Biol. 141, 53 (2003)CrossRefGoogle Scholar
  46. 46.
    Aghajan, H.K., Khalaj, B.H., Kailath, T.: Estimation of multiple 2D uniform motions by sensor array processing techniques. Presented at the image and video processing II, San Jose (1994) (unpublished)Google Scholar
  47. 47.
    Y.C. Guo, H.E. Savage, F. Liu, S.P. Schantz, P.P. Ho, R.R. Alfano, Subsurface tumor progression investigated by noninvasive optical second harmonic tomography. Proc. Natl. Acad. Sci. USA 96, 10854 (1999)CrossRefADSGoogle Scholar
  48. 48.
    E. Brown, T. McKee, E. diTomaso, A. Pluen, B. Seed, Y. Boucher, R.K. Jain, Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation. Nat. Med. 9, 796 (2003)CrossRefGoogle Scholar
  49. 49.
    P. Wilder-Smith, K. Osann, N. Hanna, N. El Abbadi, M. Brenner, D.D.V. Messadi, T. Krasieva, In vivo multiphoton fluorescence imaging: a novel approach to oral malignancy. Lasers Surg. Med. 35, 96–103 (2004)CrossRefGoogle Scholar
  50. 50.
    D.A. Dombeck, M. Blanchard-Desce, W.W. Webb, Optical recording of action potentials with second-harmonic generation microscopy. J. Neurosci. 24, 999 (2004)CrossRefGoogle Scholar
  51. 51.
    R. Leitgeb, C.K. Hitzenberger, A.F. Fercher, M. Kulhavy, Performance of fourier domain vs. time domain optical coherence tomography. Opt. Express 11, 889–894 (2003)CrossRefADSGoogle Scholar
  52. 52.
    K. Konig, T.W. Becker, P. Fisher, I. Riemann, K.J. Halbhuber, Pulse-length dependence of cellular response to intense near-infrared laser pulses in multiphoton microscopes. Opt. Lett. 24, 113–115 (1999)CrossRefADSGoogle Scholar
  53. 53.
    B.M. Kim, J. Eichler, K.M. Reiser, A.M. Rubenchik, L.B. Dasilva, Collagen structure and nonlinear susceptibility: effects of heat, glycation, and enzymatic cleavage on second harmonic signal intensity. Lasers Surg. Med. 27, 329–335 (2000)CrossRefGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2015

Authors and Affiliations

  1. 1.The Edwards Life Sciences Center for Advanced Cardiovascular TechnologyBeckman Laser InstituteIrvineUSA
  2. 2.Department of Electrical and Computer EngineeringUniversity of British ColumbiaVancouverCanada
  3. 3.Department of Biomedical Engineering, Beckman Laser InstituteUniversity of California IrvineIrvineUSA

Personalised recommendations