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^-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.

Keywords: CARS; Raman; coherent Raman; deuterium; isotopes.

FIGURE 1

Raman scattering coefficients of the C–H stretch vibrations (around 3000 cm⁻¹) and corresponding C–D stretch vibrations (around 2200 cm⁻¹) for ethane^{[ 17 ]} (a), ethene^{[ 21 ]} (b) and benzene^{[ 22 ]} (c), versus deuteration number. The open symbols are measured data; the closed symbols are results of a fit by a bond‐polarizability model. Lines are fits proportional to bond number. The ratio ϱ between the scattering coefficient per bond for C–H relative to C–D corresponding to the fits is given

FIGURE 2

Raman scattering intensities^{[ 23 ]} of water (H₂O, blue) and heavy water (D₂O, magenta) at 23°C using 488‐nm excitation. The isotropic part I _α is shown as solid line, and the anisotropic part I _γ as dashed line. The intensities scaled by a factor (ω ₀ − ω)⁻⁴ ω ⁻¹ are shown as thin lines

FIGURE 3

Scaled Raman scattering intensity R from pure SA and D4‐SA crystals. Spectra are normalized to the area of their C=O peak at 1660 cm⁻¹. The peak area over the C–H range (2700–3200 cm⁻¹) is 4.35 for SA, and 0.14 for SA‐D4 due to the O–H vibrations, while over the C–D range (1950–2450 cm⁻¹) the peak area is 3.39 for SA‐D4 (not including the N₂ peak area). The inset shows representative transmission images of the investigated SA and D4‐SA crystals

FIGURE 4

Scaled Raman scattering intensity R from oleic acid (OA) and D17‐OA. Spectra are normalized to the area of the (C=O, C=C) peak at 1660 cm⁻¹ over the range 1600–1750 cm⁻¹. The peak area over the C–H range (2600–3200 cm⁻¹) is 18.5 for OA and 9.37 for D17‐OA, while over the C–D range (1950–2450 cm⁻¹), the peak area is 9.08 for D17‐OA (not including the N₂ peak area)

FIGURE 5

Scaled Raman scattering intensity R from linoleic acid (LA) and D11‐LA. Spectra are normalized to the area of the (C=O, C=C) peak at 1660 cm⁻¹ over the range 1600–1750 cm⁻¹. The peak area over the C–H range (2600–3200 cm⁻¹) is 5.27 for LA and 3.13 for D11‐LA, while over the C–D range (1950–2450 cm⁻¹), the peak area is 1.56 for D11‐LA (not including the N₂ peak area)

FIGURE 6

Coherent anti‐Stokes Raman scattering (CARS) ratio $|\overline{\chi }{|}^2$ (black), and retrieved real (blue) and imaginary (red) part of the normalized CARS susceptibility $\overline{\chi }$, of water H₂O (thin lines) and heavy water D₂O (thick lines), at 20°C

FIGURE 7

As Figure 6, for SA (thin lines) and D4‐SA (thick lines)

FIGURE 8

As Figure 6, for OA (thin lines) and D17‐OA (thick lines)

FIGURE 9

As Figure 6, for LA (thin lines) and D11‐LA (thick lines)