Abstract
Elucidation of the molecular mechanisms regulating lipid storage and metabolism is essential for mitigating excess adiposity and obesity, which has been associated with increased prevalence of severe pathological conditions such as cardiovascular disorders and type II diabetes, worldwide. However, imaging fatty acid distribution and dynamics in vivo, at the cellular or organismal level is challenging. We developed a label-free method for visualizing lipid depositions in vivo, based on third harmonic generation (THG) microscopy. THG imaging requires a single pulsed-laser light source, alleviating the technical challenges of implementing coherent anti-Stokes Raman scattering spectroscopy (CARS) to detect fat stores in living cells. We demonstrate that THG can be used to efficiently and reliably visualize lipid droplets in Caenorhabditis elegans. Thus, THG microscopy offers a versatile alternative to fluorescence and dye-based approaches for lipid biology research.
Highlights
Visualization and monitoring of lipid depositions in living organisms is of critical importance for the study of the molecular mechanisms regulating fatty acid metabolism
Lipids are preferentially imaged in a semi-quantitative manner by Coherent Anti-stokes Raman Scattering (CARS) and Stimulated Raman Scattering (SRS), due to peakintensity signals generated by CH2, C-C, C = C and C = O, aliphatic chemical groups that are abundant in fatty acids
To overcome the limitations of dye-based and CARS/SRS microscopy, we developed a system for label-free imaging of lipids in vivo, based on Third Harmonic Generation (THG), by modifying a readily available Two-Photon Excited Fluorescence (TPEF) microscope (Figure 1a; Materials and Methods)
Summary
Visualization and monitoring of lipid depositions in living organisms is of critical importance for the study of the molecular mechanisms regulating fatty acid metabolism. To overcome the limitations of dye-based and CARS/SRS microscopy, we developed a system for label-free imaging of lipids in vivo, based on Third Harmonic Generation (THG), by modifying a readily available Two-Photon Excited Fluorescence (TPEF) microscope (Figure 1a; Materials and Methods). To evaluate the sensitivity and specificity of THG microscopy, we imaged lipid depositions in live C. elegans animals.
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