Mr. Dale Boorman Profile

Dale Boorman

at Cardiff Univ

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Proceedings Article | 4 March 2019 Paper

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

Dale Boorman, Iestyn Pope, Wolfgang Langbein, Steve Hood, Paola Borri, Peter Watson

Proceedings Volume 10890, 1089005 (2019) https://doi.org/10.1117/12.2509280

KEYWORDS: Molecules, Raman spectroscopy, Chemical analysis, CARS tomography, Microscopy, Microscopes, Hyperspectral imaging, Chemical species, Data analysis, Luminescence

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Coherent anti-Stokes Raman scattering (CARS) microscopy utilises intrinsic vibrational resonances of molecules to drive inelastic scattering of light, and thus eradicates the need for exogenous fluorescent labelling, whilst providing high-resolution three-dimensional images with chemical specificity. Replacement of hydrogen atoms with deuterium presents a labelling strategy that introduces minimal change to compound structure yet is compatible with CARS due to an induced down-shift of the CH2 peak into a region of the Raman spectrum which does not contain contributions 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 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.

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