title: Optimization of Picrosirius red staining protocol to determine collagen fiber orientations in vaginal and uterine cervical tissues by Mueller polarized microscopy.
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.
authors.affiliation: 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.
authors.forename: André
authors.initials: A
authors.lastname: Nazac
authors.affiliation: Laboratoire D'optique Et Biosciences, Ecole Polytechnique, CNRS, INSERM U696, Palaiseau, 91128, France.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Bancelin
authors.affiliation: Department of Anatomopathology, Bicêtre Hospital, Le Kremlin Bicêtre, France.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Teig
authors.affiliation: Laboratoire De Physique Des Interfaces Et Des Couches Minces (LPICM), Ecole Polytechnique, CNRS, Palaiseau, 91128, France.
authors.forename: Bicher Haj
authors.initials: BH
authors.lastname: Ibrahim
authors.affiliation: Department of Gynecology and Obstetrics, Bicêtre Hospital, Le Kremlin Bicêtre, France.|Paris XI University, Orsay, France.
authors.forename: Hervé
authors.initials: H
authors.lastname: Fernandez
authors.affiliation: Laboratoire D'optique Et Biosciences, Ecole Polytechnique, CNRS, INSERM U696, Palaiseau, 91128, France.
authors.forename: Marie-Claire
authors.initials: MC
authors.lastname: Schanne-Klein
authors.affiliation: Laboratoire De Physique Des Interfaces Et Des Couches Minces (LPICM), Ecole Polytechnique, CNRS, Palaiseau, 91128, France.
authors.forename: Antonello
authors.initials: A
authors.lastname: De Martino
mesh_terms: D001391:Azo Compounds / Q000032:analysis / Q000737:chemistry*; D003094:Collagen / Q000737:chemistry* / Q000502:physiology; D005260:Female; D006651:Histocytochemistry; D006801:Humans; D008859:Microscopy, Polarization / Q000379:methods*; D014599:Uterus / Q000737:chemistry*; D014621:Vagina / Q000737:chemistry*
chemical_list: D001391:Azo Compounds; C009798:C.I. direct red 80; D003094:Collagen
keywords: Mueller polarimetry; collagen; connective tissue; histological staining; multiphoton microscopy; polarized microscopy