Second Harmonic Generation Correlation Spectroscopy for Nanocrystal Characterization

Abstract

Many early trials at protein crystallization produce large amounts of sub-diffraction limited crystals. These nanocrystalline showers are challenging to quantitatively characterize by conventional optical methods; however, they can offer important indicators for improving crystallization conditions. Additionally, the advent and availability of ultrafast X-ray free-electron lasers now 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, limiting their broader utility. Second harmonic generation correlation spectroscopy (SHG-CS) is being developed to address this key characterization need. Under tight focus and high laser intensity, highly-ordered (crystalline) material lacking inversion symmetry; including the vast majority of protein crystals but not simple salt crystals, amorphous protein, solvents, etc.; allow for second harmonic generation, the frequency doubling of light. This provides a way to selectively track crystalline protein particles in solution. The size of the particles can be determined by taking advantage of the fact that the particles diffuse 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 autocorrelation. Although in principle, SHG-CS is similar to fluorescence correlation spectroscopy, practical challenges arising from the use of high average power (>100 mW) require constant motion of the beam. The coherence of SHG can also present unique challenges and potential opportunities compared to fluorescence-based correlation methods.