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 Sikora
  • David Scheiner
  • Cornelia Betschart
  • Daniele Perucchini
  • José María Mateos
  • Anthony di Natale
  • Daniel Fink
  • Caroline Maake
Original Article
First Online:
Received:
Accepted:
  • 270 Downloads

Abstract

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.

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.

Results

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.

Conclusions

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.

Keywords

Multiphoton microscopy Collagen Elastin Vagina Pelvic organ prolapse 
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Notes

Acknowledgments

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

Conflicts of interest

None.

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Copyright information

© The International Urogynecological Association 2014

Authors and Affiliations

  • Michal Sikora
    • 1
  • 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

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