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 . © 2015 by John Wiley & Sons, Inc.