title: Simultaneous Raman microspectroscopy and fluorescence imaging of bone mineralization in living zebrafish larvae.
abstract: Confocal Raman microspectroscopy and fluorescence imaging are two well-established methods providing functional insight into the extracellular matrix and into living cells and tissues, respectively, down to single molecule detection. In living tissues, however, cells and extracellular matrix coexist and interact. To acquire information on this cell-matrix interaction, we developed a technique for colocalized, correlative multispectral tissue analysis by implementing high-sensitivity, wide-field fluorescence imaging on a confocal Raman microscope. As a proof of principle, we study early stages of bone formation in the zebrafish (Danio rerio) larvae because the zebrafish has emerged as a model organism to study vertebrate development. The newly formed bones were stained using a calcium fluorescent marker and the maturation process was imaged and chemically characterized in vivo. Results obtained from early stages of mineral deposition in the zebrafish fin bone unequivocally show the presence of hydrogen phosphate containing mineral phases in addition to the carbonated apatite mineral. The approach developed here opens significant opportunities in molecular imaging of metabolic activities, intracellular sensing, and trafficking as well as in vivo exploration of cell-tissue interfaces under (patho-)physiological conditions.
authors.affiliation: Department of Biomaterials, Max Planck Institute of Colloids and Interfaces, Potsdam, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Bennet
authors.affiliation: Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel.
authors.forename: A
authors.initials: A
authors.lastname: Akiva
authors.affiliation: Department of Biomaterials, Max Planck Institute of Colloids and Interfaces, Potsdam, Germany.
authors.forename: D
authors.initials: D
authors.lastname: Faivre
authors.affiliation: Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel.
authors.forename: G
authors.initials: G
authors.lastname: Malkinson
authors.affiliation: Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel.
authors.forename: K
authors.initials: K
authors.lastname: Yaniv
authors.affiliation: Institute for Molecular Biology, Medizinische Hochschule Hannover, Hannover, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Abdelilah-Seyfried
authors.affiliation: Department of Biomaterials, Max Planck Institute of Colloids and Interfaces, Potsdam, Germany.
authors.forename: P
authors.initials: P
authors.lastname: Fratzl
authors.affiliation: Department of Biomaterials, Max Planck Institute of Colloids and Interfaces, Potsdam, Germany. Electronic address: masic@mpikg.mpg.de.
authors.forename: A
authors.initials: A
authors.lastname: Masic
mesh_terms: D000818:Animals; D002113:Calcification, Physiologic; D007814:Larva / Q000502:physiology; D008856:Microscopy, Fluorescence / Q000379:methods*; D061848:Optical Imaging / Q000379:methods*; D013059:Spectrum Analysis, Raman / Q000379:methods*; D015027:Zebrafish