Multiplex coherent anti-Stokes Raman scattering microscopy on lipid droplets in HeLa cells

Lipid droplets have become a major research topic recently, as they are found to be involved in obesity related diseases. Most of this research has been focused on the localization of the proteins playing a role in lipid droplet formation or breakdown. The role of different lipid species however remains unclear because it is difficult to distinguish different fatty acids with the present microscopy techniques. Coherent Anti-Stokes Raman scattering (CARS) is the non-linear analogue of spontaneous Raman scattering. Multiplex CARS microscopy can provide quantitative, chemical and physical information, making it an excellent tool to study the composition and thermodynamic phase of lipid droplets. To investigate the potential of CARS in this field, we have incubated HeLa cells with four different fatty acids, varying in saturation. The fatty acids were internalized by the cells and stored as lipid droplets, which we imaged with multiplex CARS microscopy. We were able to distinguish either of the fatty acids as such in lipid droplets inside the cells. Furthermore, we found that solid phase fatty acids were fluidized when present in lipid droplets. This illustrates the potential of CARS microscopy to elucidate the possible role of the chemistry of fatty acids in lipid droplet regulation.
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