Second harmonic generation for probing nanoscopic order within biological systems
Abstract
Many early protein crystallization trials produce large amounts of small, sometimes sub-diffraction limited crystals. These small crystals are challenging to quantitatively characterize by conventional optical methods; however, may still be good candidates for X-ray diffraction (XRD) or may offer important indicators for improving crystallization conditions. Additionally, the advent of ultrafast X-ray free-electron lasers allows single-pulse diffraction from individual protein nanocrystals for structure determination. However, these and other applications of nanocrystals currently suffer major bottlenecks in sample characterization. Second harmonic generation (SHG) can be a powerful tool for providing this characterization. An instrument has been developed for integrating SHG with a synchrotron An image with SHG takes only seconds to locate a protein crystal, even for very small crystals and crystals in turbid media such as lipidic cubic phase which would be difficult to impossible to locate with conventional imaging. This reduces or eliminates the need for X-ray raster scanning, which wastes valuable X-ray time and can damage the crystal before diffraction can be acquired. Integration with the synchrotron beam line allows a sample to be mounted on the goniometer, imaged, crystal centered and ready for diffraction in just a few minutes. This instrument has enabled direct observation of the local electric fields produced from photoelectron–hole separation upon exposure of soft materials to hard X-rays during diffraction analysis under cryogenic conditions. These results provide direct experimental observables capable of validating simulations of X-ray induced damage within soft materials. In addition, X-ray induced SHG provides a convenient method for rapidly identifying regions previously exposed to or affected by X-ray exposure. In crystallization conditions that produce nanocrystals SHG correlation spectroscopy (SHG-CS) is being developed to address the key characterization need. Second harmonic generation provides a way to selectively track crystalline protein particles in solution. The size of the particles can be determined by taking advantage of particles diffusing through solution. The amount of time spent in a particular pixel along with the size of the pixel allows diffusion constants to be extracted by temporal correlation.
Degree
Ph.D.
Advisors
Simpson, Purdue University.
Subject Area
Biochemistry
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