Volume 78, Issue 8 p. 723-730
Research Article

Optimization of Picrosirius red staining protocol to determine collagen fiber orientations in vaginal and uterine cervical tissues by Mueller polarized microscopy

André Nazac

Corresponding Author

André Nazac

Department of Gynecology and Obstetrics, Bicêtre Hospital, Le Kremlin Bicêtre, France

Laboratoire De Physique Des Interfaces Et Des Couches Minces (LPICM), Ecole Polytechnique, CNRS, Palaiseau, 91128 France

Correspondence to: A. Nazac; Department of Gynecology, Bicêtre Hospital, 78 Rue General Leclerc, 94270 Le Kremlin Bicêtre, France. E-mail: [email protected]Search for more papers by this author
Stéphane Bancelin

Stéphane Bancelin

Laboratoire D'optique Et Biosciences, Ecole Polytechnique, CNRS, INSERM U696, Palaiseau, 91128 France

Search for more papers by this author
Benjamin Teig

Benjamin Teig

Department of Anatomopathology, Bicêtre Hospital, Le Kremlin Bicêtre, France

Search for more papers by this author
Bicher Haj Ibrahim

Bicher Haj Ibrahim

Laboratoire De Physique Des Interfaces Et Des Couches Minces (LPICM), Ecole Polytechnique, CNRS, Palaiseau, 91128 France

Search for more papers by this author
Hervé Fernandez

Hervé Fernandez

Department of Gynecology and Obstetrics, Bicêtre Hospital, Le Kremlin Bicêtre, France

Paris XI University, Orsay, France

Search for more papers by this author
Marie-Claire Schanne-Klein

Marie-Claire Schanne-Klein

Laboratoire D'optique Et Biosciences, Ecole Polytechnique, CNRS, INSERM U696, Palaiseau, 91128 France

Search for more papers by this author
Antonello De Martino

Antonello De Martino

Laboratoire De Physique Des Interfaces Et Des Couches Minces (LPICM), Ecole Polytechnique, CNRS, Palaiseau, 91128 France

Search for more papers by this author
First published: 12 June 2015
Citations: 15

REVIEW EDITOR: Prof. Alberto Diaspro

ABSTRACT

Polarized microscopy provides unique information on anisotropic samples. In its most complete implementation, namely Mueller microscopy, this technique is well suited for the visualization of fibrillar proteins orientations, with collagen in the first place. However, the intrinsic optical anisotropy of unstained tissues has to be enhanced by Picrosirius Red (PR) staining to enable Mueller measurements. In this work, we compared the orientation mapping provided by Mueller and second harmonic generation (SHG) microscopies on PR stained samples of vaginal and uterine cervix tissues. SHG is a multiphoton technique that is highly specific to fibrillar collagen, and was taken as the “gold standard” for its visualization. We showed that Mueller microscopy can be safely used to determine collagen orientation in PR stained cervical tissue. In contrast, in vaginal samples, Mueller microscopy revealed orientations not only of collagen but also of other anisotropic structures. Thus PR is not fully specific to collagen, which necessitates comparison to SHG microscopy in every type of tissue. In addition to this study of PR specificity, we determined the optimal values of the staining parameters. We found that staining times of 5 min, and sample thicknesses of 5 µm were sufficient in cervical and vaginal tissues. Microsc. Res. Tech. 78:723–730, 2015. © 2015 Wiley Periodicals, Inc.