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

Abstract

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.

Dual coherent anti‐Stokes Raman scattering microscopy across the vibrational spectrum enabled by single pump optically synchronised oscillators and spectral focusing

“…To demonstrate the imaging of a three‐component mixture with resonances in the silent region, a sample containing an oil, water and heavy water reverse emulsion (see Section 2.2.2) was measured. To target water, deuterated water and oil vibrational frequencies, the pump beam was tuned to 831 nm to address with the fixed Stokes beam the deuterated water C–D stretch [21] at 2400 . Both delay lines were calibrated according to the procedure described in Appendix .…”

“…Simultaneously, the pump and the variable Stokes beam at 1110 nm were addressing a minimum of the oil signal at 3080 cm À1 due to the destructive interference of the resonant signal and the non-resonant background. [21] The resulting S CH is shown in (B) and combined with (A) in the red-blue colour overlay in (C). The variable Stokes beam and the delay line were then tuned to 1072 nm to address the oil resonance at 2850 cm À1 , and the resulting simultaneously acquired S SR and S CH are given in (D) and (E), respectively, with a red-green colour overlay shown in (F).…”

Hyperspectral CARS microscopy and quantitative unsupervised analysis of deuterated and non-deuterated fatty acid storage in human cells

“…CARS intensity ratios were then calculated by dividing the background-corrected CARS intensity by the corresponding non-resonant CARS intensity measured in glass under the same excitation and detection conditions. The phase-corrected Kramers-Kronig (PCKK) method was used to retrieve from the CARS intensity ratio the complex CARS third-order susceptibility (in units of the non-resonant susceptibility of glass 54 ), which is linear in the concentration of chemical components. Non-negative matrix factorization applied to the imaginary part and the average real part of the susceptibility with an additional concentration constraint (FSC 3 ) was used to obtain susceptibility spectra of separate chemical components and their volume concentrations in vol/vol units.…”

“…53 Notably, in these previous hyperspectral CARS studies, we did not explore the use of Raman tags. Very recently, we studied the quantitative CRS susceptibility of deuterated compounds, 54 showing that the susceptibility of the stretch vibrations approximately scales with their frequency under isotope substitution.…”

ECONOS/ECW 2020: In memoriam Marcus Motzkus

“…With his frequent invited talks and contributions, he truly shaped the meetings and delivered memorable highlights. Therefore, it is no surprise that so many groups associated with ECONOS/ECW immediately committed to contribute to this special issue, [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] which happens to be the largest collection of papers in this annual series in more than a decade.…”