Multimodal nonlinear optical imaging of Caenorhabditis elegans with multiplex coherent anti-Stokes Raman scattering, third-harmonic generation, second-harmonic generation, and two-photon excitation fluorescence

Multimodal nonlinear optical microscopy was applied to the nematode Caenorhabditis elegans. Transgenic worms expressing the yellow fluorescent protein variant Venus underwent analyses. The lipid and protein distribution was clearly visualized using multiplex coherent anti-Stokes Raman scattering, second-harmonic generation (SHG), third-harmonic generation, and two-photon excitation fluorescence (TPEF). The TPEF and SHG signals were found to overlap, which allowed the identification of the ventral nerve cord. These findings demonstrate the powerful potential of multimodal nonlinear optical microscopy to reveal C. elegans microstructures in vivo.