title: Label-free, three-dimensional multiphoton microscopy of the connective tissue in the anterior vaginal wall.
abstract: OBJECTIVE
  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.
authors.affiliation: Department of Gynecology, University Hospital Zurich, Frauenklinikstrasse 10, 8091, Zurich, Switzerland, michal.sikora@usz.ch.
authors.forename: Michal
authors.initials: M
authors.lastname: Sikora
authors.forename: David
authors.initials: D
authors.lastname: Scheiner
authors.forename: Cornelia
authors.initials: C
authors.lastname: Betschart
authors.forename: Daniele
authors.initials: D
authors.lastname: Perucchini
authors.forename: José María
authors.initials: JM
authors.lastname: Mateos
authors.forename: Anthony
authors.initials: A
authors.lastname: di Natale
authors.forename: Daniel
authors.initials: D
authors.lastname: Fink
authors.forename: Caroline
authors.initials: C
authors.lastname: Maake
mesh_terms: D003094:Collagen; D003238:Connective Tissue / Q000033:anatomy & histology*; D004549:Elastin; D005109:Extracellular Matrix; D005260:Female; D006652:Histological Techniques; D006801:Humans; D021621:Imaging, Three-Dimensional; D018613:Microscopy, Confocal / Q000379:methods*; D036641:Microscopy, Fluorescence, Multiphoton; D014621:Vagina / Q000033:anatomy & histology*
chemical_list: D003094:Collagen; D004549:Elastin