Detection of early osteogenic commitment in primary cells using Raman spectroscopy

Major challenges in the development of novel implant surfaces for artificial joints include osteoblast heterogeneity and the lack of a simple and sensitive in vitro assay to measure early osteogenic responses. Raman spectroscopy is a label-free, non-invasive and non-destructive vibrational fingerprinting optical technique that is increasingly being applied to detect biochemical changes in cells. In this study Raman spectroscopy has been used to obtain bone cell-specific spectral signatures and to identify any changes therein during osteoblast commitment and differentiation of primary cells in culture. Murine calvarial osteoblasts (COBs) were extracted and cultured and studied by Raman spectroscopy over a 14 day culture period. Distinct osteogenic Raman spectra were identified after 3 days of culture with strong bands detected for mineral: phosphate ν3 (1030 cm−1) and B-type carbonate (1072 cm−1), DNA (782 cm−1) and collagen matrix (CH2 deformation at 1450 cm−1) and weaker phosphate bands (948 and 970 cm−1). Early changes were detected by Raman spectroscopy compared to a standard enzymatic alkaline phosphatase (ALP) assay and gene expression analyses over this period. Proliferation of COBs was confirmed by fluorescence intensity measurements using the Picogreen dsDNA reagent. Changes in ALP levels were evident only after 14 days of culture and mRNA expression levels for ALP, Col1a1 and Sclerostin remained constant during the culture period. Sirius red staining for collagen deposition also revealed little change until day 14. In contrast Raman spectroscopy revealed the presence of amorphous calcium phosphate (945–952 cm−1) and carbonated apatite (957–962 cm−1) after only 3 days in culture and octacalcium phosphate (970 cm−1) considered a transient mineral phase, was detected after 5 days of COBs culture. PCA analysis confirmed clear separation between time-points. This study highlights the potential of Raman spectroscopy to be utilised for the early and specific detection of proliferation and differentiation changes in primary cultures of bone cells

Silicon-nitride waveguides for on-chip Raman spectroscopy

The evanescent tail of the guided modes can efficiently excite Raman active molecules located in the cladding of a waveguide. Similarly, a significant fraction of the total emitted Stokes power is evanescently coupled to the same mode. Further, the enhancement effects inherent to the waveguide, alongside with the long interaction length, lead to an increased light-matter interaction, resulting in a higher sensitivity as required by spectroscopic applications, especially in the context of Raman spectroscopy. We calculate the spontaneous Raman scattering efficiency as a function of siliconnitride strip waveguide dimensions and show that under typical conditions, the overall efficiency is approximately two orders of magnitude higher than in confocal configuration in the free space. We also report the experimental demonstration of the use of silicon-nitride based photonic waveguides in a lab-on-a-chip context for Raman spectroscopy. To the best of our knowledge, this is the first demonstration of Raman spectroscopy using photonic waveguides

Application of Raman Microspectroscopic and Raman imaging techniques for cell biological studies

Raman spectroscopy is being used to study biological molecules for some three decades now. Thanks to continuing advances in instrumentation more and more applications have become feasible in which molecules are studied in situ, and this has enabled Raman spectroscopy to enter the realms of biomedicine and cell biology [1-5].\ud Here we will describe some of the recent work carried out in our laboratory, concerning studies of human white blood cells and further instrumentational developments

Band-edge Bilayer Plasmonic Nanostructure for Surface Enhanced Raman Spectroscopy

Spectroscopic analysis of large biomolecules is critical in a number of applications, including medical diagnostics and label-free biosensing. Recently, it has been shown that Raman spectroscopy of proteins can be used to diagnose some diseases, including a few types of cancer. These experiments have however been performed using traditional Raman spectroscopy and the development of the Surface enhanced Raman spectroscopy (SERS) assays suitable for large biomolecules could lead to a substantial decrease in the amount of specimen necessary for these experiments. We present a new method to achieve high local field enhancement in surface enhanced Raman spectroscopy through the simultaneous adjustment of the lattice plasmons and localized surface plasmon polaritons, in a periodic bilayer nanoantenna array resulting in a high enhancement factor over the sensing area, with relatively high uniformity. The proposed plasmonic nanostructure is comprised of two interacting nanoantenna layers, providing a sharp band-edge lattice plasmon mode and a wide-band localized surface plasmon for the separate enhancement of the pump and emitted Raman signals. We demonstrate the application of the proposed nanostructure for the spectral analysis of large biomolecules by binding a protein (streptavidin) selectively on the hot-spots between the two stacked layers, using a low concentration solution (100 nM) and we successfully acquire its SERS spectrum

Raman Topography and Strain Uniformity of Large-Area Epitaxial Graphene

We report results from two-dimensional Raman spectroscopy studies of large-area epitaxial graphene grown on SiC. Our work reveals unexpectedly large variation in Raman peak position across the sample resulting from inhomogeneity in the strain of the graphene film, which we show to be correlated with physical topography by coupling Raman spectroscopy with atomic force microscopy. We report that essentially strain free graphene is possible even for epitaxial graphene.Comment: 10 pages, 3 figure

Intercalation of Hydrotalcites with Hexacyanoferrate(II) and (III)-a ThermoRaman Spectroscopic Study

Raman spectroscopy using a hot stage indicates that the intercalation of hexacyanoferrate(II) and (III) in the interlayer space of a Mg,Al hydrotalcites leads to layered solids where the intercalated species is both hexacyanoferrate(II) and (III). Raman spectroscopy shows that depending on the oxidation state of the initial hexacyanoferrate partial oxidation and reduction takes place upon intercalation. For the hexacyanoferrate(III) some partial reduction occurs during synthesis. The symmetry of the hexacyanoferrate decreases from Oh existing for the free anions to D3d in the hexacyanoferrate interlayered hydrotalcite complexes. Hot stage Raman spectroscopy reveals the oxidation of the hexacyanoferrate(II) to hexacyanoferrate(III) in the hydrotalcite interlayer with the removal of the cyanide anions above 250 °C. Thermal treatment causes the loss of CN ions through the observation of a band at 2080 cm-1. The hexacyanoferrate (III) interlayered Mg,Al hydrotalcites decomposes above 150 °C

FAST CARS: Engineering a Laser Spectroscopic Technique for Rapid Identification of Bacterial Spores

Airborne contaminants, e.g., bacterial spores, are usually analyzed by time consuming microscopic, chemical and biological assays. Current research into real time laser spectroscopic detectors of such contaminants is based on e.g. resonant Raman spectroscopy. The present approach derives from recent experiments in which atoms and molecules are prepared by one (or more) coherent laser(s) and probed by another set of lasers. The connection with previous studies based on "Coherent Anti-Stokes Raman Spectroscopy" (CARS) is to be noted. However generating and utilizing maximally coherent oscillation in macromolecules having an enormous number of degrees of freedom is much more challenging. This extension of the CARS technique is called FAST CARS (Femtosecond Adaptive Spectroscopic Techniques for Coherent Anti-Stokes Raman Spectroscopy), and the present paper proposes and analyses ways in which it could be used to rapidly identify pre-selected molecules in real time.Comment: 43 pages, 21 figures; replacement with references added. Submitted to the Proceedings of National Academy of Science

A review on applications of two-dimensional materials in surface enhanced Raman spectroscopy

Two-dimensional (2D) materials, such as graphene and MoS2, have been attracting wide interest in surface enhancement Raman spectroscopy. This perspective gives an overview of recent developments in 2D materials' application in surface enhanced Raman spectroscopy. This review focuses on the applications of using bare 2D materials and metal/2D material hybrid substrate for Raman enhancement. The Raman enhancing mechanism of 2D materials will also be discussed. The progress covered herein shows great promise for widespread adoption of 2D materials in SERS application.Comment: 14 pages, 7 Figure