International Urogynecology Journal

, Volume 26, Issue 5, pp 685–691 | Cite as

Label-free, three-dimensional multiphoton microscopy of the connective tissue in the anterior vaginal wall

  • Michal SikoraEmail author
  • David Scheiner
  • Cornelia Betschart
  • Daniele Perucchini
  • José María Mateos
  • Anthony di Natale
  • Daniel Fink
  • Caroline Maake
Original Article


Introduction and hypothesis

Multiphoton microscopy (MPM) is a nonlinear, high-resolution laser scanning technique and a powerful approach for analyzing the spatial architecture within tissues. To demonstrate the potential of this technique for studying the extracellular matrix of the pelvic organs, we aimed to establish protocols for the detection of collagen and elastin in the vagina and to compare the MPM density of these fibers to fibers detected using standard histological methods.


Samples of the anterior vaginal wall were obtained from nine patients undergoing a hysterectomy or cystocele repair. Samples were shock frozen, fixed with formaldehyde or Thiel’s solution, or left untreated. Samples were imaged with MPM to quantify the amount of collagen and elastin via second harmonic generation and autofluorescence, respectively. In six patients, sample sections were also histologically stained and imaged with brightfield microscopy. The density of the fibers was quantified using the StereoInvestigator and Cavalieri software.


With MPM, collagen and elastin could be visualized to a depth of 100 μm, and no overlap of signals was detected. The different tissue processing protocols used did not result in significantly different fiber counts after MPM. MPM-based fiber quantifications are comparable to those based on conventional histological stains. However, MPM provided superior resolution, particularly of collagen fibers.


MPM is a robust, rapid, and label-free method that can be used to quantify the collagen and elastin content in thick specimens of the vagina. It is an excellent tool for future three-dimensional studies of the extracellular matrix in patients with pelvic organ prolapse.


Multiphoton microscopy Collagen Elastin Vagina Pelvic organ prolapse 



The authors would like to thank Theresa Lehmann and Charlotte Burger for technical assistance with the tissue processing and histological staining.

Conflicts of interest



  1. 1.
    Kerkhof MH, Hendriks L, Brölmann HA (2009) Changes in connective tissue in patients with pelvic organ prolapse–a review of the current literature. Int Urogynecol J Pelvic Floor Dysfunct 20(4):461–474. doi: 10.1007/s00192-008-0737-1 CrossRefPubMedGoogle Scholar
  2. 2.
    De Landsheere L, Munaut C, Nusgens B, Maillard C, Rubod C, Nisolle M, Cosson M, Foidart JM (2013) Histology of the vaginal wall in women with pelvic organ prolapse: a literature review. Int Urogynecol J 24(12):2011–2020. doi: 10.1007/s00192-013-2111-1 CrossRefPubMedGoogle Scholar
  3. 3.
    Abraham T, Hogg J (2010) Extracellular matrix remodeling of lung alveolar walls in three dimensional space identified using second harmonic generation and multiphoton excitation fluorescence. J Struct Biol 171(2):189–196. doi: 10.1016/j.jsb.2010.04.006 CrossRefPubMedGoogle Scholar
  4. 4.
    Zhu X, Lin L, Yu H, Zhuo S, Chen J, Liu J, Wang Y (2012) Visualization of epidermal and dermal alteration in papulonodular mucinosis by multiphoton microscopy. Scanning 35(1):22–27. doi: 10.1002/sca.21031 CrossRefPubMedGoogle Scholar
  5. 5.
    Phillippi JA, Green BR, Eskay MA, Kotlarczyk MP, Hill MR, Robertson AM, Watkins SC, Vorp DA, Gleason TG (2014) Mechanism of aortic medial matrix remodeling is distinct in patients with bicuspid aortic valve. J Thorac Cardiovasc Surg 147(3):1056–1064. doi: 10.1016/j.jtcvs.2013.04.028 CrossRefPubMedCentralPubMedGoogle Scholar
  6. 6.
    Ustione A, Piston DW (2011) A simple introduction to multiphoton microscopy. J Microsc 243(3):221–226. doi: 10.1111/j.1365-2818.2011.03532.x CrossRefPubMedGoogle Scholar
  7. 7.
    Friedl P, Wolf K, von Andrian UH, Harms G (2007) Biological second and third harmonic generation microscopy. Curr Protoc Cell Biol Chapter 4:Unit 4.15. doi: 10.1002/0471143030.cb0415s34
  8. 8.
    Schenke-Layland K (2008) Non-invasive multiphoton imaging of extracellular matrix structures. J Biophotonics 1(6):451–462. doi: 10.1002/jbio.200810045 CrossRefPubMedCentralPubMedGoogle Scholar
  9. 9.
    Theer P, Hasan MT, Denk W (2003) Two-photon imaging to a depth of 1000 microm in living brains by use of a Ti:Al2O3 regenerative amplifier. Opt Lett 28(12):1022–1024CrossRefPubMedGoogle Scholar
  10. 10.
    Chen XN, Nadiarynkh O, Plotnikov S, Campagnola PJ (2012) Second harmonic generation microscopy for quantitative analysis of collagen fibrillar structure. Nat Protoc 7(4):654–669. doi: 10.1038/nprot.2012.009 CrossRefPubMedCentralPubMedGoogle Scholar
  11. 11.
    Tong PL, Qin J, Cooper CL, Lowe PM, Murrell DF, Kossard S, Ng LG, Roediger B, Weninger W, Haass NK (2013) A quantitative approach to histopathological dissection of elastin-related disorders using multiphoton microscopy. Br J Dermatol 169(4):869–879. doi: 10.1111/bjd.12430 CrossRefPubMedGoogle Scholar
  12. 12.
    Chen J, Zhu X, Xu Y, Tang Y, Xiong S, Zhuo S, Chen J (2014) Stereoscopic visualization and quantification of auricular cartilage regeneration in rabbits using multiphoton microscopy. Scanning 36(5):540–546. doi: 10.1002/sca.21153 CrossRefPubMedGoogle Scholar
  13. 13.
    Cui JZ, Tehrani AY, Jett KA, Bernatchez P, van Breemen C, Esfandiarei M (2014) Quantification of aortic and cutaneous elastin and collagen morphology in Marfan syndrome by multiphoton microscopy. J Struct Biol 187(3):242–253. doi: 10.1016/j.jsb.2014.07.003 CrossRefPubMedGoogle Scholar
  14. 14.
    Martin TP, Norris G, McConnell G, Currie S (2013) A novel approach for assessing cardiac fibrosis using label-free second harmonic generation. Int J Cardiovasc Imaging 29(8):1733–1740. doi: 10.1007/s10554-013-0270-2 CrossRefPubMedGoogle Scholar
  15. 15.
    Bump RC, Mattiasson A, Bø K, Brubaker LP, DeLancey JO, Klarskov P, Shull BL, Smith AR (1996) The standardization of terminology of female pelvic organ prolapse and pelvic floor dysfunction. Am J Obstet Gynecol 175(1):10–17CrossRefPubMedGoogle Scholar
  16. 16.
    Thiel W (1992) The preservation of the whole corpse with natural color. Ann Anat 174(3):185–195CrossRefPubMedGoogle Scholar
  17. 17.
    Böck P (ed) (1989) Romeis Mikroskopische Technik, 17th edn. Urban und Schwarzenberg, MunichGoogle Scholar
  18. 18.
    Howard CV, Reed MG (eds) (1998) Unbiased stereology. BIOS Scientific Publishers, OxfordGoogle Scholar
  19. 19.
    Gundersen HJ, Jensen EB, Kiêu K, Nielsen J (1999) The efficiency of systematic sampling in stereology–reconsidered. J Microsc 193(Pt 3):199–211CrossRefPubMedGoogle Scholar
  20. 20.
    Peloquin J, Huynh J, Williams RM, Reinhart-King CA (2011) Indentation measurements of the subendothelial matrix in bovine carotid arteries. J Biomech 44(5):815–821. doi: 10.1016/j.jbiomech.2010.12.018 CrossRefPubMedGoogle Scholar
  21. 21.
    Chen H, Liu Y, Slipchenko MN, Zhao X, Cheng JX, Kassab GS (2011) The layered structure of coronary adventitia under mechanical load. Biophys J 101(11):2555–2562. doi: 10.1016/j.bpj.2011.10.043 CrossRefPubMedCentralPubMedGoogle Scholar
  22. 22.
    Riemann I, Le Harzic R, Mpoukouvalas K, Heimann A, Kempski O, Charalampaki P (2012) Sub-cellular tumor identification and markerless differentiation in the rat brain in vivo by multiphoton microscopy. Lasers Surg Med 44(9):719–725. doi: 10.1002/lsm.22079 CrossRefPubMedGoogle Scholar
  23. 23.
    Yu Y, Lee AM, Wang H, Tang S, Zhao J, Lui H, Zeng H (2012) Imaging-guided two-photon excitation-emission-matrix measurements of human skin tissues. J Biomed Opt 17(7):077004. doi: 10.1117/1.JBO.17.7.077004 CrossRefPubMedGoogle Scholar
  24. 24.
    Chen AC, McNeilly C, Liu AP, Flaim CJ, Cuttle L, Kendall M, Kimble RM, Shimizu H, McMillan JR (2011) Second harmonic generation and multiphoton microscopic detection of collagen without the need for species specific antibodies. Burns 37(6):1001–1009. doi: 10.1016/j.burns.2011.03.013 CrossRefPubMedGoogle Scholar
  25. 25.
    Zhu X, Zhuo S, Zheng L, Jiang X, Chen J, Lin B (2011) Quantification of scar margin in keloid different from atrophic scar by multiphoton microscopic imaging. Scanning 33(4):195–200. doi: 10.1002/sca.20230 PubMedGoogle Scholar
  26. 26.
    Meek K (1981) The use of glutaraldehyde and tannic acid to preserve reconstituted collagen for electron microscopy. Histochemistry 73(1):115–120CrossRefPubMedGoogle Scholar
  27. 27.
    Collins JS, Goldsmith TH (1981) Spectral properties of fluorescence induced dy glutaraldehyde fixation. J Histochem Cytochem 29(3):411–414CrossRefPubMedGoogle Scholar
  28. 28.
    Adur J, Pelegati VB, de Thomaz AA, D’Souza-Li L, Assunção Mdo C, Bottcher-Luiz F, Andrade LA, Cesar CL (2012) Quantitative changes in human epithelial cancers and osteogenesis imperfecta disease detected using nonlinear multicontrast microscopy. J Biomed Opt 17(8):081407–081401. doi: 10.1117/1.JBO.17.8.081407 CrossRefPubMedGoogle Scholar
  29. 29.
    Fritze O, Schleicher M, König K, Schenke-Layland K, Stock U, Harasztosi C (2010) Facilitated noninvasive visualization of collagen and elastin in blood vessels. Tissue Eng Part C Methods 16(4):705–710. doi: 10.1089/ten.TEC.2009.0309 CrossRefPubMedGoogle Scholar
  30. 30.
    Alperin M, Moalli PA (2006) Remodeling of vaginal connective tissue in patients with prolapse. Curr Opin Obstet Gynecol 18(5):544–550CrossRefPubMedGoogle Scholar
  31. 31.
    Lin SY, Tee YT, Ng SC, Chang H, Lin P, Chen GD (2007) Changes in the extracellular matrix in the anterior vagina of women with or without prolapse. Int Urogynecol J Pelvic Floor Dysfunct 18(1):43–48. doi: 10.1007/s00192-006-0090-1 CrossRefPubMedGoogle Scholar
  32. 32.
    Meijerink AM, van Rijssel RH, van der Linden PJ (2012) Tissue composition of the vaginal wall in women with pelvic organ prolapse. Gynecol Obstet Invest 75(1):21–27. doi: 10.1159/000341709 CrossRefPubMedGoogle Scholar
  33. 33.
    Boulesteix T, Pena AM, Pagès N, Godeau G, Sauviat MP, Beaurepaire E, Schanne-Klein MC (2006) Micrometer scale ex vivo multiphoton imaging of unstained arterial wall structure. Cytometry A 69(1):20–26. doi: 10.1002/cyto.a.20196 CrossRefPubMedGoogle Scholar
  34. 34.
    Le TT, Langohr IM, Locker MJ, Sturek M, Cheng JX (2007) Label-free molecular imaging of atherosclerotic lesions using multimodal nonlinear optical microscopy. J Biomed Opt 12(5):054007. doi: 10.1117/1.2795437 CrossRefPubMedCentralPubMedGoogle Scholar
  35. 35.
    Schenke-Layland K, Stock UA, Nsair A, Xie J, Angelis E, Fonseca CG, Larbig R, Mahajan A, Shivkumar K, Fishbein MC, MacLellan WR (2009) Cardiomyopathy is associated with structural remodelling of heart valve extracellular matrix. Eur Heart J 30(18):2254–2265. doi: 10.1093/eurheartj/ehp267 CrossRefPubMedCentralPubMedGoogle Scholar

Copyright information

© The International Urogynecological Association 2014

Authors and Affiliations

  • Michal Sikora
    • 1
    Email author
  • David Scheiner
    • 1
  • Cornelia Betschart
    • 1
  • Daniele Perucchini
    • 1
  • José María Mateos
    • 3
  • Anthony di Natale
    • 2
  • Daniel Fink
    • 1
  • Caroline Maake
    • 2
  1. 1.Department of GynecologyUniversity Hospital ZurichZurichSwitzerland
  2. 2.Institute of AnatomyUniversity of ZurichZurichSwitzerland
  3. 3.Center for Microscopy and Image AnalysisUniversity of ZurichZurichSwitzerland

Personalised recommendations