Second order nonlinear optical imaging of chiral crystals (SONICC) has been developed for analyzing protein crystallization trials. A brief overview of the origin of second harmonic generation (SHG), the physical process underlying SONICC, is given to explain the selectivity, based on order, and sensitivity, based on background suppression. These characteristics are demonstrated by comparing SHG signal with two photon excited fluorescence (TPEF) images of green fluorescent protein (GFP). The second order dependence of incident peak optical power also makes SONICC relatively insensitive to optical scatter. This feature is demonstrated in the imaging of membrane protein crystals in highly turbid lipidic mesophases. In this case, low background SONICC images can be automatically analyzed more reliably that expert analysis of traditional optical imaging methods. SONICC was also applied to locating protein crystals that were mounted in loops and cryogenically frozen for x-ray diffraction analysis. Based on solved structures from x-ray diffraction studies, SONICC did not cause detectable damage to the protein crystals that were imaged at cryogenic temperatures. Finally, methods were developed to extend the linear dynamic range in SONICC measurements by statistical treatment of photomultiplier tube (PMT) voltage responses and through direct digitization of those voltages. These methods will allow for quantitation over the entire dynamic range of a PMT and will lead to quantitative applications of SONICC like crystallization kinetic studies or polarization dependent measurements.
展开▼
