In situ Raman imaging of osteoblastic mineralization Aya Hashimoto, a Liang-da Chiu, a Keigo Sawada, b Tomohiko Ikeuchi, a Katsumasa Fujita, a Masahide Takedachi, b Yoshinori Yamaguchi, c * Satoshi Kawata, a,c Shinya Murakami b and Eiichi Tamiya a,c Hydroxyapatite (HA) is synthesized at early stages of bone formation by osteoblasts. Nondestructive observation of early stages of osteoblastic mineralization provides crucial information for biological mechanism of bone formation. Raman microscopy serves as an ideal tool to observe the osteoblastic mineralization process because it shows the chemical information of the sample at a minimally invasive level. In addition, HA is a marker for osteoblastic mineralization, and HA Raman signal is strong enough to identify mineralized spots in osteoblasts. In this research, we visualized the distribution of HA in cultured mouse osteoblasts by Raman imaging and observed the location of the mineralized spots in the culture. We monitored HA Raman signal from osteoblast culture for 3 days after administrating the osteogenic differentiation medium and observed Raman signal associated with HA. We identified mineralized spots of KUSA-A1 by Raman imaging constructed from the distribution of HA Raman signal. We successfully visualized the distribution of the mineralized spots in the culture of KUSA-A1. We compared our Raman images with Alizarin red S staining assay, which was a conventional method to evaluate the mineralization process. Raman imaging of the KUSA-A1 culture visualized the mineralized spots more accurately than Alizarin red S staining assay. Raman imaging of HA serves as a powerful tool to identify the mineralized spots in an in vitro culture of osteogenic lineage cells. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: Raman imaging; hydroxyapatite (HA); osteoblast; mineralization; matrix vesicle (MV) Introduction Mineralization is main function of osteoblasts. Osteoblasts undergo various differentiation processes, such as the synthesis of collagen, the expression of alkaline phosphatase, and the biogenesis of matrix vesicle (MV). Through these processes, osteoblasts calcify surrounding extracellular matrix (ECM) and finally form bone tissue. [1–9] Osteoblastic mineralization, which is the early stage of bone formation, is initiated by forming hydroxyapatite (HA) seed crystals in MVs. The HA seed crystals are released into the extracellular fluid through the MV membrane. The released seed crystals attach on extracellular collagen fibers, and the HA seed crystals grow by taking Ca 2+ and PO 4 3À in the extracellular fluid. These HA crystal depositions become mineralized nodules gradually. [10–12] Thus, the HA distribution in the cell culture indicates the sites that undergo osteoblastic mineralization. The distribution of mineralized nodules in the culture of KUSA-A1, which is a mouse mesenchymal stem cell line, is heterogeneous. It has been reported that macroscopic studies reveal that various biomolecules, for example, type I collagen, alkaline phosphatase, osteonectin, osteopontin, bone sialoprotein, and osteocalcin, are involved in the process of osteoblastic mineralization. To understand the exact mineralization mechanism, it is essential to know the concentration, location, and timing of expression of those contributing biomolecules with subcellular microscopic time-lapse observation of the mineralized spots. Therefore, exact interpretation of the mineralization process requires subcellular level time-lapse observation at the same location with the quantitative information of the biomolecules involved in the mineralization process. Detecting HA has been performed by staining phosphate (PO 4 3À ) or calcium (Ca 2+ ) with staining reagents, such as von Kossa [13] and Alizarin red S (ARS). [14,15] Both von Kossa and ARS staining assay greatly contributes to understanding bone formation. [12–14] These staining assays are inexpensive, requires no special instrumentation, and are reliable for examining the mineralization process at a macroscopic scale. Although these staining assays roughly determines the area that mineralization has occurred, the staining assays are impossible to specify the position of the HA crystals that appear in the sample. Furthermore, because the staining techniques are destructive techniques, they are unable to follow up the osteoblastic mineralization process. To understand fully the mineralization process, it is necessary to monitor mineralized * Correspondence to: Yoshinori Yamaguchi, Photonics Advanced Research Center, Graduate School of Engineering, Osaka University, Suita, Japan. E-mail: yoshi.yamaguchi@ap.eng.osaka-u.ac.jp a Department of applied physics, Graduate School of Engineering Osaka University, Suita, Japan b Department of Periodontology, Graduate School of Dentistry Osaka University, Suita, Japan c Photonics Advanced Research Center, Graduate School of Engineering Osaka University, Suita, Japan Abbreviation: TC, tissue culture; PBS, phosphate buffered saline; ECM, extracel- lular matrix; MV, matrix vesicle; FOV, field of view; HA, hydroxyapatite; FCS, fetal calf serum; αMEM, alpha Modified Eagle Minimum Essential Medium; CCD, charge-coupled device. J. Raman Spectrosc. 2014, 45, 157–161 Copyright © 2014 John Wiley & Sons, Ltd. Research article Received: 17 May 2013 Revised: 31 October 2013 Accepted: 3 December 2013 Published online in Wiley Online Library: 16 January 2014 (wileyonlinelibrary.com) DOI 10.1002/jrs.4438 157