Estimating the helical pitch angle of amylopectin in starch using polarization second harmonic generation microscopy

Starch granules are among the brightest natural second harmonic generation (SHG) converters. They basically consist of amylose and amylopectin molecules and the source of the SHG signal is still undetermined. In the present study we perform polarization sensitive SHG (PSHG) imaging of wheat starch granules and we fit the SHG signal variation of each pixel of the PSHG images into a generalized biophysical model. By assuming that the SHG source molecule is a helix with cylindrical symmetry along its long axis, the model extracts the helical pitch angle of the SHG source for every pixel of the image. By displaying the pixel histogram representing this helical pitch angle we found a highly peaked histogram with maximum at theta(e) = 36.1 degrees and a width of Delta theta(e) = 9.3 degrees. This pitch angle corresponds to the strand of the parallel double helical structure, called amylopectin (as measured by a small angle x-ray scattering technique). This demonstrates amylopectin (and not amylose) as the source of SHG in starch. According to our knowledge, this is the first estimation of the amylopectin's pitch angle in starch using PSHG.