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Abstract: We present a new method, second harmonic generation (SHG) imaging for the study of starch structure. SHG imaging can provide the structural organization and molecular orientation information of bio-tissues without centrosymmetry. In recent years, SHG has proven its capability in the study of crystallized bio-molecules such as collagen and myosin. Starch, the most important food source and a promising future energy candidate, has, for a decade, been shown to exhibit strong SHG response. By comparing SHG intensity from different starch species, we first identified that the SHG-active molecule is amylopectin, which accounts for the crystallinity in starch granules. With the aid of SHG polarization anisotropy, we extracted the complete χ (2) tensor of amylopectin, which reflects the underlying molecular details. Through χ (2) tensor analysis, three-dimensional orientation and packing symmetry of amylopectin are determined. The helical angle of the double-helix in amylopectin is also deduced from the tensor, and the value corresponds well to previous X-ray studies, further verifying amylopectin as SHG source. It is noteworthy that the nm-sized structure of amylopectin inside a starch granule can be determined by this far-field optical method with 1-μm excitation wavelength. Since SHG is a relatively new tool for plant research, a detailed understanding of SHG in starch structure will be useful for future high-resolution imaging and quantitative analyses for food/energy applications. [Copyright &y& Elsevier]