Quantification of the nonlinear susceptibility of the hydrogen and deuterium stretch vibration for biomolecules in coherent Raman micro‐spectroscopy

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(−1) for C–H into the silent region around 2100 cm(−1) 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.