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IR super-resolution imaging of the cross section of the rachis of an avian feather was carried out by using a vibrational sum-frequency generation (VSFG) detected IR microscope with a sub-micrometer spatial resolution. In the YYX polarization combination, we clearly observed strong signals in the entire region of the rachis at the amide I vibration of β-keratin. On the other hand, the signal disappears from most of the cross section in the XXY polarization combination. Because the VSFG imaging detects the signal only from the interface,