Deuterium labelling is increasingly used in coherent Raman imaging of complex systems, such as biological cells and tissues, to improve chemical specificity. Nevertheless, quantitative coherent Raman susceptibility spectra for deuterated compounds have not been previously reported. Interestingly, it is expected theoretically that -D stretch vibrations have a Raman susceptibility lower than -H stretch vibrations, with the area of their imaginary part scaling with their wavenumber, which is shifted from around 2900 cm À1 for C-H into the silent region around 2100 cm À1 for C-D. Here, we report quantitative measurements of the nonlinear susceptibility of water, succinic acid, oleic acid, linoleic acid and deuterated isoforms. We show that the -D stretch vibration has indeed a lower area, consistent with the frequency reduction due to the doubling of atomic mass from hydrogen to deuterium. This finding elucidates an important trade-off between chemical specificity and signal strength in the adoption of deuterium labelling as an imaging strategy for coherent Raman microscopy.

Note: This paper is part of the JCP Special Topic on Chemical Imaging.

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 CH 2 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 (FSC 3), 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 FSC 3 analysis enabled identification of the intracellular location of the exogenously applied deuterated lipid against the chemical background of the cell. Through application of FSC 3 analysis, deuterium-labelling may provide a powerful technique for imaging small molecules which are poorly suited to conventional fluorescence techniques.