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


Sikora, Michal; Scheiner, David; Betschart, Cornelia; Perucchini, Daniele; Mateos, José María; di Natale, Anthony; Fink, Daniel; Maake, Caroline (2015). Label-free, three-dimensional multiphoton microscopy of the connective tissue in the anterior vaginal wall. International Urogynecology Journal and Pelvic Floor Dysfunction, 26(5):685-691.

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.

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.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Institute of Anatomy
04 Faculty of Medicine > Center for Microscopy and Image Analysis
04 Faculty of Medicine > University Hospital Zurich > Clinic for Gynecology
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Date:2015
Deposited On:05 Feb 2015 08:35
Last Modified:05 Apr 2016 18:54
Publisher:Springer
ISSN:0937-3462
Publisher DOI:https://doi.org/10.1007/s00192-014-2571-y
PubMed ID:25421935

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