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We present our work using deuterated oleic acid to optimise setup of an in-house-developed multimodal, multiphoton, laser-scanning microscope for precise identification of carbon-deuterium-associated peaks within the silent region of the Raman spectrum. Application of the data analysis procedure, factorisation into susceptibilities and concentrations of chemical components (FSC3), enables the identification and quantitative spatial resolution of specific deuterated chemical components within a hyperspectral CARS image. Full hyperspectral CARS datasets were acquired from HeLa cells incubated with either deuterated or non-deuterated oleic acid, and subsequent FSC3 analysis enabled identification of the intracellular location of the exogenously applied deuterated lipid against the chemical background of the cell. Through application of FSC3 analysis, deuterium-labelling may provide a powerful technique for imaging small molecules which are poorly suited to conventional fluorescence techniques.
Imaging lipids in living mammalian oocytes and early embryos by coherent Raman scattering microscopy
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