Lipid-cell interactions in human monocytes investigated by doubly-resonant coherent anti-Stokes Raman scattering microscopy.

We demonstrate that doubly-resonant coherent anti-Stokes Raman scattering can provide enhanced and highly specific contrast for molecules containing unique Raman-active small molecular groups. This combination provides contrast for molecules that can otherwise be difficult to discriminate by Raman spectroscopy. Here, human monocytes were incubated with either deuterated oleic acid or 17-octadecynoic acid (a fatty acid with an end terminal acetylene group). The carbon-deuterium stretching vibration of the deuterated fatty acid, as well as the unique alkyne stretching vibration of the alkyne-containing fatty acid, were used to provide contrast for these exogenous free fatty acids. The combination of these unique modes with the common aliphatic carbon-hydrogen stretching vibration inherent to all fatty acid allowed for doubly-resonant detection of these unique molecules and enabled us to detect the presence of these lipids in areas within a cell where each molecular resonance by itself did not generate sufficient signal.