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Second harmonic generation correlation spectroscopy (SHG-CS) is demonstrated as a new approach to protein nanocrystal characterization. A novel line-scanning approach was performed to enable autocorrelation analysis without sample damage from the intense incident beam. An analytical model for autocorrelation was developed, which includes a correction for the optical scattering forces arising when focusing intense, infrared beams. SHG-CS was applied to the analysis of BaTiO3 nanoparticles ranging from 200 to ∼500 nm and of photosystem I nanocrystals. A size distribution was recovered for each sample and compared with the size histogram measured by scanning electron microscopy (SEM). Good agreement was observed between the two independent measurements. The intrinsic selectivity of the second-order nonlinear optical process provides SHG-CS with the ability to distinguish well ordered nanocrystals from conglomerates and amorphous aggregates. Combining the recovered distribution of particle diameters with the histogram of measured SHG intensities provides the inherent hyperpolarizability per unit volume of the SHG-active nanoparticles. Simulations suggest that the SHG activity per unit volume is likely to exhibit relatively low sensitivity to the subtle distortions within the lattice that contribute to resolution loss in X-ray diffraction, but high sensitivity to the presence of multi-domain crystals.

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