Imaging of Rhodopsin Crystals with Two-Photon Microscopy

Two-photon microscopy has been shown to be an invaluable tool for detecting and monitoring protein crystallization trials and characterizing membrane protein crystals. This imaging method has proven especially useful for rhodopsin, because of the dependence of rhodopsin's fluorescence spectra on the isomerization state of its intrinsic chromophore (retinylidene) and, as such, it can provide additional information about the identity and functional state of rhodopsin in crystals. Here, we describe the acquisition of images and two-photon excitation and emission spectra using a commercial two-photon microscope, along with detailed instructions for the handling of rhodopsin crystals and specific examples of rhodopsin data.