Molecular organization of crystalline β-carotene in carrots determined with polarization-dependent second and third harmonic generation microscopy.

Polarization-in, polarization-out (PIPO) second harmonic generation (SHG) and third harmonic generation (THG) microscopy was used to study the crystalline organization of β-carotene molecules within individual aggregates contained in the chromoplasts of orange carrots in vivo. Multimodal PIPO SHG and PIPO THG studies of the aggregates revealed one dominant SHG and THG dipole signifying that β-carotene molecules are oriented along a single axis. Three-dimensional visualization of the orientation of β-carotene molecules with respect to the aggregate axis was also performed with both microscopy modalities and revealed organization of the aggregates as ribbon-like structures consisting of twists and folds. Therefore, PIPO SHG and PIPO THG microscopy provides information on the crystalline organization and the orientation of ordered biological structures in vivo where multimodal polarization dependent SHG and THG investigations are particularly advantageous as both noncentrosymmetric and centrosymmetric crystalline organizations can be probed.