Organization of Astaxanthin within Oil Bodies of Haematococcus pluvialis Studied with Polarization-Dependent Harmonic Generation Microscopy

Nonlinear optical microscopy was used to image the localization of astaxanthin accumulation in the green alga, Haematococcus pluvialis. Polarization-in, polarization-out (PIPO) second harmonic generation (SHG) and third harmonic generation (THG) microscopy was applied to study the crystalline organization of astaxanthin molecules in light-stressed H. pluvialis in vivo. Since astaxanthin readily forms H- and J-aggregates in aqueous solutions, PIPO THG studies of astaxanthin aggregates contained in red aplanospores were compared to PIPO THG of in vitro self-assembled H- and J-aggregates of astaxanthin. The PIPO THG data clearly showed an isotropic organization of astaxanthin in red aplanospores of H. pluvialis. This is in contrast to the highly anisotropic organization of astaxanthin in synthetic H- and J-aggregates, which showed to be uniaxial. Since carotenoids in vitro preferentially form H- and J-aggregates, but in vivo form a randomly organized structure, this implies that astaxanthin undergoes a different way of packing in biological organisms, which is either due to the unique physical environment of the alga or is controlled enzymatically.