Imaging of chemical and physical state of individual cellular lipid droplets using multiplex CARS microscopy

Cellular lipid droplets are organelles that carry out important functions such as the regulated storage and release of cholesterol and fatty acids (FAs). Lipid droplets therefore play a key role in both healthy metabolism and metabolic disorders such as obesity, type 11 diabetes, and atherosclerosis. Hence, there has been much interest in understanding the chemical (lipid) composition and physical state (lipid packing) of lipid droplets. Coherent anti-Stokes Raman Scattering (CARS) microscopy has proven to be a useful tool in lipid droplet research because of its ability to visualize lipid droplets in a label-free manner. Multiplex CARS microscopy in spectroscopy mode has been particularly useful: In conjunction with appropriate spectral processing, it allows one to obtain the local Raman response with submicron spatial resolution. Thus, multiplex CARS microscopy provides rapid quantitative information and allows quantitative imaging of the chemistry (level of acyl unsaturation) and physical state (acyl chain order) of individual lipid droplets. We discuss the different vibrational markers that may be used to characterize the lipid droplet properties and demonstrate that while the C-H stretch of unsaturated =C-H groups is a useful marker of the level of acyl unsaturation in vitro, it fails in vivo. Moreover, we present here results on two different cell lines - adipocytes and HeLa cells - with distinct lipid droplet characteristics, both in terms of lipid droplet size and dependence of the lipid droplet composition on the incubation medium.