| We have investigated the molecular origins of second order optical nonlinear effects in proteins with sum frequency generation vibrational spectroscopy. We have focused our investigation on two protein structures, poly-gamma-benzyl-L-glutamate (PBLG), a synthetic single helical protein, and collagen, an extracellular triple helical protein. We have found that the second order nonlinearity in PBLG arises from the collective contribution of amide A and amide I groups that are organized in a noncentrosymmetric manner by hydrogen bonding. In contrast, in the case of collagen, the second order nonlinear effects are due to (1) achiral contribution from the methylene groups associated with Fermi resonance between the fundamental symmetric stretch and the bending overtone of methylene, and (2) a chiral contribution by an intramolecular helical arrangement of carbonyl and peptide groups.;We have used polarization modulated second harmonic generation and sum frequency generation imaging to investigate the spatial distribution of molecules in protein films and biological structures. We introduce a new image processing approach, spectral moment invariants, which quantifies texture in images. We then use the spectral moment invariants to discriminate between normal and damaged collageneous tissue imaged by polarization modulated second harmonic generation imaging. A detailed experimental study has been performed on spinal disk injuries, and it has been shown that the quantification of tissue disorder with spectral moment invariants correlates well with the degree of tissue deterioration. This finding demonstrates the potential clinical application of polarization modulated second harmonic imaging to detect extracellular related diseases. |
