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Fibrosis pp 409-425 | Cite as

Probing Collagen Organization: Practical Guide for Second-Harmonic Generation (SHG) Imaging

  • Riccardo Cicchi
  • Francesco S. Pavone
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1627)

Abstract

Second-harmonic generation (SHG) microscopy is a powerful microscopy technique for imaging collagen and other biological molecules using a label-free approach. SHG microscopy offers the advantages of a nonlinear imaging modality together with those ones of a coherent technique. These features make SHG microscopy the ideal tool for imaging collagen at high resolution and for characterizing its organization at various hierarchical levels. Considering that collagen organization plays a crucial role in fibrosis and in its development, it would be beneficial for the researcher working in the field of fibrosis to have a manual listing crucial points to be considered when imaging collagen using SHG microscopy. This chapter provides an answer to this demand with state-of-the-art protocols, methods, and laboratory tips related to SHG microscopy. We also discuss advantages and limitations of the use of SHG for studying fibrosis.

Key words

SHG microscopy Collagen Polarization scanning Forward backward Detection Numerical aperture Ti:sapphire laser 

Notes

Acknowledgments

We acknowledge funding from the Italian Ministry for Education, University and Research in the framework of the Flagship Project NANOMAX, from Tuscany Region and EU FP7 BiophotonicsPlus projects “LITE” (Laser Imaging of The Eye) and “LighTPatcH” (Led Technology in Photo Haemostasis), from the European Union Seventh Framework Programme (FP7/2007–2013) under grant agreement number 284464 and number 604102 (Human Brain Project), from the Italian Ministry of Health (GR-2011-02349626), from Fondazione Pisa, and from Ente Cassa di Risparmio di Firenze. Part of this work was performed in the frame of the Proof of Concept Studies for the ESFRI research infrastructure project Euro-BioImaging at the PCS facility LENS.

References

  1. 1.
    Sheppard CJR, Gannaway J, Kompfner R et al (1977) The scanning harmonic optical microscope. IEEE J Quantum Electron 13E:100D. doi: 10.1109/JQE.1977.1069615 Google Scholar
  2. 2.
    Gannaway JN, Sheppard CJR (1978) Second-harmonic imaging in the scanning optical microscope. Opt Quant Electron 10(5):435–439. doi: 10.1007/bf00620308 CrossRefGoogle Scholar
  3. 3.
    Denk W, Strickler JH, Webb WW (1990) Two-photon laser scanning fluorescence microscopy. Science 248(4951):73–76. doi: 10.1126/science.2321027 CrossRefPubMedGoogle Scholar
  4. 4.
    Dunn AK, Wallace VP, Coleno M et al (2000) Influence of optical properties on two-photon fluorescence imaging in turbid samples. Appl Opt 39(7):1194–1201. doi: 10.1364/AO.39.001194 CrossRefPubMedGoogle Scholar
  5. 5.
    Helmchen F, Denk W (2005) Deep tissue two-photon microscopy. Nat Methods 2(12):932–940. doi: 10.1038/nmeth818 CrossRefPubMedGoogle Scholar
  6. 6.
    Gu M, Gan XS, Kisteman A et al (2000) Comparison of penetration depth between two-photon excitation and single-photon excitation in imaging through turbid tissue media. Appl Phys Lett 77(10):1551–1553. doi: 10.1063/1.1308059 CrossRefGoogle Scholar
  7. 7.
    Oheim M, Beaurepaire E, Chaigneau E et al (2001) Two-photon microscopy in brain tissue: parameters influencing the imaging depth. J Neurosci Methods 111(1):29–37. doi: 10.1016/S0165-0270(01)00438-1 CrossRefPubMedGoogle Scholar
  8. 8.
    Beaurepaire E, Oheim M, Mertz J (2001) Ultra-deep two-photon fluorescence excitation in turbid media. Opt Commun 188(1–4):25–29. doi: 10.1016/S0030-4018(00)01156-1 CrossRefGoogle Scholar
  9. 9.
    Sheppard CJR, Gu M (1990) Image formation in two-photon fluorescence microscopy. Optik 86(3):104–110Google Scholar
  10. 10.
    Nakamura O (1999) Fundamental of two-photon microscopy. Microsc Res Tech 47(3):165–171. doi:10.1002/(SICI)1097-0029(19991101)47:3<165::AID-JEMT2>3.0.CO;2-DCrossRefPubMedGoogle Scholar
  11. 11.
    Xu C, Zipfel W, Shear JB et al (1996) Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy. Proc Natl Acad Sci U S A 93(20):10763–10768. doi: 10.1073/pnas.93.20.10763 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Zipfel WR, Williams RM, Christie R et al (2003) Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation. Proc Natl Acad Sci U S A 100(12):7075–7080. doi: 10.1073/pnas.0832308100 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Zipfel WR, Williams RM, Webb WW (2003) Nonlinear magic: multiphoton microscopy in the biosciences. Nat Biotechnol 21(11):1369–1377. doi: 10.1038/nbt899 CrossRefPubMedGoogle Scholar
  14. 14.
    Centonze VE, White JG (1998) Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging. Biophys J 75(4):2015–2024. doi: 10.1016/S0006-3495(98)77643-X CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Fine S, Hansen WP (1971) Optical second harmonic generation in biological systems. Appl Opt 10(10):2350–2353. doi: 10.1364/AO.10.002350 CrossRefPubMedGoogle Scholar
  16. 16.
    Mohler W, Millard AC, Campagnola PJ (2003) Second harmonic generation imaging of endogenous structural proteins. Methods 29(1):97–109. doi: 10.1016/S1046-2023(02)00292-X CrossRefPubMedGoogle Scholar
  17. 17.
    Roth S, Freund I (1979) Second-harmonic generation in collagen. J Chem Phys 70(4):1637–1643. doi: 10.1063/1.437677 CrossRefGoogle Scholar
  18. 18.
    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(4):693–712. doi: 10.1016/S0006-3495(86)83510-X CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Boulesteix T, Beaurepaire E, Sauviat MP et al (2004) Second-harmonic microscopy of unstained living cardiac myocytes: measurements of sarcomere length with 20-nm accuracy. Opt Lett 29(17):2031–2033. doi: 10.1364/OL.29.002031 CrossRefPubMedGoogle Scholar
  20. 20.
    Both M, Vogel M, Friedrich O et al (2004) Second harmonic imaging of intrinsic signals in muscle fibers in situ. J Biomed Opt 9(5):882–892. doi: 10.1117/1.1783354 CrossRefPubMedGoogle Scholar
  21. 21.
    Plotnikov SV, Millard AC, Campagnola PJ et al (2006) Characterization of the myosin-based source for second-harmonic generation from muscle sarcomeres. Biophys J 90(2):693–703. doi: 10.1529/biophysj.105.071555 CrossRefPubMedGoogle Scholar
  22. 22.
    Llewellyn ME, Barretto RP, Delp SL et al (2008) Minimally invasive high-speed imaging of sarcomere contractile dynamics in mice and humans. Nature 454(7205):784–788. doi: 10.1038/nature07104 PubMedPubMedCentralGoogle Scholar
  23. 23.
    Nucciotti V, Stringari C, Sacconi L et al (2010) Probing myosin structural conformation in vivo by second-harmonic generation microscopy. Proc Natl Acad Sci U S A 107(17):7763–7768. doi: 10.1073/pnas.0914782107
  24. 24.
    Dombeck DA, Kasischke KA, Vishwasrao HD et al (2003) Uniform polarity microtubule assemblies imaged in native brain tissue by second-harmonic generation microscopy. Proc Natl Acad Sci U S A 100(12):7081–7086. doi: 10.1073/pnas.0731953100 CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Yeh AT, Nassif N, Zoumi A et al (2002) Selective corneal imaging using combined second-harmonic generation and two-photon excited fluorescence. Opt Lett 27(23):2082–2084. doi: 10.1364/OL.27.002082 CrossRefPubMedGoogle Scholar
  26. 26.
    Han M, Giese G, Bille J (2005) Second harmonic generation imaging of collagen fibrils in cornea and sclera. Opt Express 13(15):5791–5797. doi: 10.1364/OPEX.13.005791 CrossRefPubMedGoogle Scholar
  27. 27.
    Matteini P, Ratto F, Rossi F et al (2009) Photothermally-induced disordered patterns of corneal collagen revealed by SHG imaging. Opt Express 17(6):4868–4878. doi: 10.1364/OE.17.004868 CrossRefPubMedGoogle Scholar
  28. 28.
    Stoller P, Kim BM, Rubenchik AM et al (2002) Polarization-dependent optical second-harmonic imaging of a rat-tail tendon. J Biomed Opt 7(2):205–214. doi: 10.1117/1.1431967 CrossRefPubMedGoogle Scholar
  29. 29.
    Theodossiou T, Rapti GS, Hovhannisyan V et al (2002) Thermally induced irreversible conformational changes in collagen probed by optical second harmonic generation and laser-induced fluorescence. Lasers Med Sci 17(1):34–41. doi: 10.1007/s101030200007 CrossRefPubMedGoogle Scholar
  30. 30.
    Zoumi A, Lu X, Kassab GS et al (2004) Imaging coronary artery microstructure using second-harmonic and two-photon fluorescence microscopy. Biophys J 87(4):2778–2786. doi: 10.1529/biophysj.104.042887 CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Cicchi R, Matthaus C, Meyer T et al (2014) Characterization of collagen and cholesterol deposition in atherosclerotic arterial tissue using non-linear microscopy. J Biophotonics 7(1–2):135–143. doi: 10.1002/jbio.201300055 CrossRefPubMedGoogle Scholar
  32. 32.
    Stoller P, Reiser KM, Celliers PM et al (2002) Polarization-modulated second harmonic generation in collagen. Biophys J 82(6):3330–3342. doi: 10.1016/S0006-3495(02)75673-7 CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Yasui T, Tohno Y, Araki T (2004) Characterization of collagen orientation in human dermis by two-dimensional second-harmonic-generation polarimetry. J Biomed Opt 9(2):259–264. doi: 10.1117/1.1644116 CrossRefPubMedGoogle Scholar
  34. 34.
    Cicchi R, Sestini S, De Giorgi V et al (2008) Nonlinear laser imaging of skin lesions. J Biophotonics 1(1):62–73. doi: 10.1002/jbio.200710003 CrossRefPubMedGoogle Scholar
  35. 35.
    Su PJ, Chen WL, Hong JB et al (2009) Discrimination of collagen in normal and pathological skin dermis through second-order susceptibility microscopy. Opt Express 17(13):11161–11171. doi: 10.1364/OE.17.011161 CrossRefPubMedGoogle Scholar
  36. 36.
    Pena AM, Fagot D, Olive C et al (2010) Multiphoton microscopy of engineered dermal substitutes: assessment of 3-D collagen matrix remodeling induced by fibroblast contraction. J Biomed Opt 15(5):056018. doi: 10.1117/1.3503411 CrossRefPubMedGoogle Scholar
  37. 37.
    Cicchi R, Kapsokalyvas D, De Giorgi V et al (2010) Scoring of collagen organization in healthy and diseased human dermis by multiphoton microscopy. J Biophotonics 3(1–2):34–43. doi: 10.1002/jbio.200910062 PubMedGoogle Scholar
  38. 38.
    Medyukhina A, Vogler N, Latka I et al (2011) Automated classification of healthy and keloidal collagen patterns based on processing of SHG images of human skin. J Biophotonics 4(9):627–636. doi: 10.1002/jbio.201100028 PubMedGoogle Scholar
  39. 39.
    Chen J, Zhuo S, Jiang X et al (2011) Multiphoton microscopy study of the morphological and quantity changes of collagen and elastic fiber components in keloid disease. J Biomed Opt 16(5):051305. doi: 10.1117/1.3569617 CrossRefPubMedGoogle Scholar
  40. 40.
    Lin SJ, Hsiao CY, Sun Y et al (2005) Monitoring the thermally induced structural transitions of collagen by use of second-harmonic generation microscopy. Opt Lett 30(6):622–624. doi: 10.1364/OL.30.000622 CrossRefPubMedGoogle Scholar
  41. 41.
    Lo W, Chang YL, Liu JS et al (2009) Multimodal, multiphoton microscopy and image correlation analysis for characterizing corneal thermal damage. J Biomed Opt 14(5):054003. doi: 10.1117/1.3213602 CrossRefPubMedGoogle Scholar
  42. 42.
    Matteini P, Cicchi R, Ratto F et al (2012) Thermal transitions of fibrillar collagen unveiled by second-harmonic generation microscopy of corneal stroma. Biophys J 103(6):1179–1187. doi: 10.1016/j.bpj.2012.07.055 CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Guo Y, Savage HE, Liu F et al (1999) Subsurface tumor progression investigated by noninvasive optical second harmonic tomography. Proc Natl Acad Sci U S A 96(19):10854–10856. doi: 10.1073/pnas.96.19.10854 CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Brown E, McKee T, diTomaso E et al (2003) Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation. Nat Med 9(6):796–800. doi: 10.1038/nm879
  45. 45.
    Provenzano PP, Eliceiri KW, Campbell JM et al (2006) Collagen reorganization at the tumor-stromal interface facilitates local invasion. BMC Med 4(1):38. doi: 10.1186/1741-7015-4-38 CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Lin SJ, Jee SH, Kuo CJ et al (2006) Discrimination of basal cell carcinoma from normal dermal stroma by quantitative multiphoton imaging. Opt Lett 31(18):2756–2758. doi: 10.1364/OL.31.002756 CrossRefPubMedGoogle Scholar
  47. 47.
    Han X, Burke RM, Zettel ML et al (2008) Second harmonic properties of tumor collagen: determining the structural relationship between reactive stroma and healthy stroma. Opt Express 16(3):1846–1859. doi: 10.1364/OE.16.001846 CrossRefPubMedGoogle Scholar
  48. 48.
    Nadiarnykh O, LaComb RB, Brewer MA et al (2010) Alterations of the extracellular matrix in ovarian cancer studied by second harmonic generation imaging microscopy. BMC Cancer 10:94. doi: 10.1186/1471-2407-10-94 CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Raja AM, Xu S, Sun W et al (2010) Pulse-modulated second harmonic imaging microscope quantitatively demonstrates marked increase of collagen in tumor after chemotherapy. J Biomed Opt 15(5):056016. doi: 10.1117/1.3497565 CrossRefPubMedGoogle Scholar
  50. 50.
    Plotnikov SV, Kenny AM, Walsh SJ et al (2008) Measurement of muscle disease by quantitative second-harmonic generation imaging. J Biomed Opt 13(4):044018. doi: 10.1117/1.2967536 CrossRefPubMedGoogle Scholar
  51. 51.
    Nadiarnykh O, Plotnikov S, Mohler WA et al (2007) Second harmonic generation imaging microscopy studies of osteogenesis imperfecta. J Biomed Opt 12(5):051805. doi: 10.1117/1.2799538 CrossRefPubMedGoogle Scholar
  52. 52.
    Lacomb R, Nadiarnykh O, Campagnola PJ (2008) Quantitative second harmonic generation imaging of the diseased state osteogenesis imperfecta: experiment and simulation. Biophys J 94(11):4504–4514. doi: 10.1529/biophysj.107.114405 CrossRefPubMedPubMedCentralGoogle Scholar
  53. 53.
    Reiser KM, Bratton C, Yankelevich DR et al (2007) Quantitative analysis of structural disorder in intervertebral disks using second harmonic generation imaging: comparison with morphometric analysis. J Biomed Opt 12(6):064019. doi: 10.1117/1.2812631 CrossRefPubMedGoogle Scholar
  54. 54.
    Tiwari N, Chabra S, Mehdi S et al (2010) Imaging of normal and pathologic joint synovium using nonlinear optical microscopy as a potential diagnostic tool. J Biomed Opt 15(5):056001. doi: 10.1117/1.3484262 CrossRefPubMedPubMedCentralGoogle Scholar
  55. 55.
    Strupler M, Pena AM, Hernest M et al (2007) Second harmonic imaging and scoring of collagen in fibrotic tissues. Opt Express 15(7):4054–4065. doi: 10.1364/OE.15.004054 CrossRefPubMedGoogle Scholar
  56. 56.
    Strupler M, Hernest M, Fligny C et al (2008) Second harmonic microscopy to quantify renal interstitial fibrosis and arterial remodeling. J Biomed Opt 13(5):054041. doi: 10.1117/1.2981830 CrossRefPubMedGoogle Scholar
  57. 57.
    Pena AM, Fabre A, Debarre D et al (2007) Three-dimensional investigation and scoring of extracellular matrix remodeling during lung fibrosis using multiphoton microscopy. Microsc Res Tech 70(2):162–170. doi: 10.1002/jemt.20400 CrossRefPubMedGoogle Scholar
  58. 58.
    Gailhouste L, Le Grand Y, Odin C et al (2010) Fibrillar collagen scoring by second harmonic microscopy: a new tool in the assessment of liver fibrosis. J Hepatol 52(3):398–406. doi: 10.1016/j.jhep.2009.12.009 CrossRefPubMedGoogle Scholar
  59. 59.
    Guilbert T, Odin C, Le Grand Y et al (2010) A robust collagen scoring method for human liver fibrosis by second harmonic microscopy. Opt Express 18(25):25794–25807. doi: 10.1364/OE.18.025794 CrossRefPubMedGoogle Scholar
  60. 60.
    Barad Y, Eisenberg H, Horowitz M et al (1997) Nonlinear scanning laser microscopy by third harmonic generation. Appl Phys Lett 70:922–924. doi: 10.1063/1.118442 CrossRefGoogle Scholar
  61. 61.
    Oron D, Yelin D, Tal E et al (2004) Depth-resolved structural imaging by third-harmonic generation microscopy. J Struct Biol 147(1):3–11. doi: 10.1016/S1047-8477(03)00125-4 CrossRefPubMedGoogle Scholar
  62. 62.
    Debarre D, Supatto W, Pena AM et al (2006) Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy. Nat Methods 3(1):47–53. doi: 10.1038/nmeth813 CrossRefPubMedGoogle Scholar
  63. 63.
    Mertz J, Moreaux L (2001) Second-harmonic generation by focused excitation of inhomogeneously distributed scatterers. Opt Commun 196:325–330. doi: 10.1016/S0030-4018(01)01403-1 CrossRefGoogle Scholar
  64. 64.
    Vanzi F, Sacconi L, Cicchi R et al (2012) Protein conformation and molecular order probed by second-harmonic-generation microscopy. J Biomed Opt 17(6):060901. doi: 10.1117/1.JBO.17.6.060901 CrossRefPubMedGoogle Scholar
  65. 65.
    Cicchi R, Vogler N, Kapsokalyvas D et al (2013) From molecular structure to tissue architecture: collagen organization probed by SHG microscopy. J Biophotonics 6(2):129–142. doi: 10.1002/jbio.201200092 CrossRefPubMedGoogle Scholar

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© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.National Institute of OpticsNational Research Council (INO-CNR)Sesto FiorentinoItaly
  2. 2.European Laboratory for Non-linear Spectroscopy (LENS)University of FlorenceSesto FiorentinoItaly
  3. 3.Department of PhysicsUniversity of FlorenceSesto FiorentinoItaly

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