title: Polychrome labeling of bone with seven different fluorochromes: enhancing fluorochrome discrimination by spectral image analysis.
abstract: Bone formation and remodeling in vivo can be assessed by polychrome labeling using calcium-binding fluorescent dyes. The number of fluorochromes, however, limits this technique due to the fact that with increasing number, fluorescent spectra inevitably overlap, which makes discrimination more difficult. In order to enhance discrimination, we performed spectral image analysis. Non-critical size defects of the femur of male Wistar rats served as a model for bone formation. Eight different fluorochromes (calcein blue, xylenol orange, calcein, alizarine complexone, doxycycline, rolitetracycline, hematoporphyrin, and BAPTA) were administered sequentially subcutaneously every third day starting at day 4 after surgery. Following, bone specimen were embedded in methylmethacrylate and analyzed by spectral image acquisition using a Sagnac type interferometer (ASI, Israel). Seven of the eight applied fluorochromes could be resolved using spectral image examination. With BAPTA, we present a new fluorochrome suitable for bone labeling. Due to the superior sensitivity of the spectral image acquisition, the thickness of the bone sections could be reduced so that 5 mum thick sections could be analyzed. Spectral decomposition and subsequent linear unmixing allows depiction of each individual fluorochrome without interference of any other, enabling a reliable and superior morphometric analysis of labeled regions.
authors.affiliation: Department of Oral and Cranio-Maxillofacial Surgery, Klinikum rechts der Isar, Technical University of Munich (TUM), Ismaninger Str. 22, D-81675 Munich, Germany. christoph.pautke@gmx.net
authors.forename: Christoph
authors.initials: C
authors.lastname: Pautke
authors.forename: Stephan
authors.initials: S
authors.lastname: Vogt
authors.forename: Thomas
authors.initials: T
authors.lastname: Tischer
authors.forename: Gabriele
authors.initials: G
authors.lastname: Wexel
authors.forename: Herbert
authors.initials: H
authors.lastname: Deppe
authors.forename: Stefan
authors.initials: S
authors.lastname: Milz
authors.forename: Matthias
authors.initials: M
authors.lastname: Schieker
authors.forename: Andreas
authors.initials: A
authors.lastname: Kolk
mesh_terms: D000818:Animals; D001842:Bone and Bones / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D008297:Male; D008856:Microscopy, Fluorescence; D051381:Rats; D017208:Rats, Wistar; D013057:Spectrum Analysis / Q000379:methods*
chemical_list: D005456:Fluorescent Dyes