Optimisation of multimodal coherent anti-Stokes Raman scattering microscopy for the detection of isotope-labelled molecules

摘要

Coherent anti-Stokes Raman scattering (CARS) microscopy utilises intrinsic vibrational resonances ofmolecules to drive inelastic scattering of light, and thus eradicates the need for exogenous fluorescent labelling, whilstproviding high-resolution three-dimensional images with chemical specificity. Replacement of hydrogen atoms withdeuterium presents a labelling strategy that introduces minimal change to compound structure yet is compatible withCARS due to an induced down-shift of the CH_2 peak into a region of the Raman spectrum which does not containcontributions from other chemical species, thus giving contrast against other cellular components.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 silentregion of the Raman spectrum. Application of the data analysis procedure, factorisation into susceptibilities andconcentrations of chemical components (FSC~3), enables the identification and quantitative spatial resolution of specificdeuterated chemical components within a hyperspectral CARS image. Full hyperspectral CARS datasets were acquiredfrom HeLa cells incubated with either deuterated or non-deuterated oleic acid, and subsequent FSC~3 analysis enabledidentification of the intracellular location of the exogenously applied deuterated lipid against the chemical background ofthe cell. Through application of FSC~3 analysis, deuterium-labelling may provide a powerful technique for imaging smallmolecules which are poorly suited to conventional fluorescence techniques.