High-Resolution Multi-Photon Imaging of Morphological Structures of Caenorhabditis elegans

In this protocol, we combine two-photon excitation fluorescence with nonlinear optical measurements to reconstruct the three-dimensional architecture of the pharyngeal region and the muscular system of the anterior and mid-body region of Caenorhabditis elegans (C. elegans). Femto-second laser pulses excite second-harmonic generation (SHG) and third-harmonic generation (THG) signals, which show detailed structural information regarding the organization of myofibrils that are arranged around the central pharynx region. The combination of two-photon excitation with SHG and THG imaging is a very powerful tool to study cell morphology, microarchitecture, and tissue arrangement in C. elegans.