Time-lapse Raman imaging of osteoblast differentiation

Abstract

Osteoblastic mineralization occurs during the early stages of bone formation. During this mineralization, hydroxyapatite (HA), a major component of bone, is synthesized, generating hard tissue. Many of the mechanisms driving biomineralization remain unclear because the traditional biochemical assays used to investigate them are destructive techniques incompatible with viable cells. To determine the temporal changes in mineralization-related biomolecules at mineralization spots, we performed time-lapse Raman imaging of mouse osteoblasts at a subcellular resolution throughout the mineralization process. Raman imaging enabled us to analyze the dynamics of the related biomolecules at mineralization spots throughout the entire process of mineralization. Here, we stimulated KUSA-A1 cells to differentiate into osteoblasts and conducted time-lapse Raman imaging on them every 4 hours for 24 hours, beginning 5 days after the stimulation. The HA and cytochrome c Raman bands were used as markers for osteoblastic mineralization and apoptosis. From the Raman images successfully acquired throughout the mineralization process, we found that β-carotene acts as a biomarker that indicates the initiation of osteoblastic mineralization. A fluctuation of cytochrome c concentration, which indicates cell apoptosis, was also observed during mineralization. We expect time-lapse Raman imaging to help us to further elucidate osteoblastic mineralization mechanisms that have previously been unobservable.

Rapid in vivo lipid/carbohydrate quantification of single microalgal cell by Raman spectral imaging to reveal salinity-induced starch-to-lipid shift

“…1. Notably, the spectrum of 2% NaCl+ 5-day N-depletion shows some other interesting features of a weak starch peak at 479 cm −1 and an obvious carotenoid spectral feature [22] (Fig. 1) with peaks at 1526 cm −1 , 1160 cm −1 .…”

Raman Mapping: Emerging Applications

“…The same authors investigated the potential for developing Ramanactivated cell sorting of individual cells [43]. Hashimoto et al [44] got information on osteoblast differentiation and mineralization mechanisms by monitoring fluctuations in the cytochrome C concentration. The above preliminary studies suggest that Raman spectroscopy has a great potential to become a leading method for stem cells investigation.…”

Markerfreie molekulare Bildgebung biologischer Zellen und Gewebe durch lineare und nichtlineare Raman‐spektroskopische Ansätze

“…Der Vergleich der Fluoreszenzbilder von Mitochondrien und der Raman‐Bilder von Cytochrom c bestätigte, dass Veränderungen in der Cytochrom‐c‐Verteilung als Freisetzung von Cytochrom c aus Mitochondrien angesehen werden können. Zeitraffer‐Raman‐Bildgebung wurde verwendet, um die osteoblastische Mineralisierung zu untersuchen . Während dieser Mineralisierung wird Hydroxylapatit, ein Hauptbestandteil des Knochens, synthetisiert.…”