Nile Red stained worms were collected as described above. For live worm imaging, worms were collected and paralyzed on the agar pad. Lipid droplets and Nile Red or GFP fluorescence were monitored using CARS and TPEF microscopy, respectively. The setup of CARS was described in the previous report.22 Briefly, two Ti-sapphire laser beams (Mira-900P and Mira-900F, Coherent, California) were synchronized by Synchro-Lock system (Coherent, California) and tuned at and to obtain CARS imaging of lipid C-H stretching mode at . The forward CARS signal (at ) is collected by a condenser (), passing through a set of bandpass filters (FF01-630/92 and FF01-590/10, Semrock, New York) and detected by a photomultiplier tube (PMT) (R7400U-02, Hamamatsu, Japan). Fluorescence signal was collected by a water immersion objective, passing through a bandpass filter (FF01-624/40 for Nile Red and FF01-512/25 for GFP, Semrock, Rochester, New York), and detected by a PMT (R3896, Hamamatsu, Japan). The background, defined as the average intensity of CARS, was subtracted from the original image for quantification of CARS intensity.15 The number, size, and CARS intensity of lipid droplets were analyzed by using the MetaMorph software version 7.5.