Intrinsic disorder and structural biology: Searching where the light isn't.

We developed the new IR super-resolution microscope by using a 4-wave mixing (4-wave), which is a third-order nonlinear optical process, and carried out the IR super-resolution imaging of the cross section of the rachis of an avian feather. We clearly observed strong signals in the entire region of the rachis at the amide I vibration of β-keratin in both of the XXYY and YYXX polarization combination. These results are different from images detected by using the vibrational sum-frequency generation (VSFG) method. While the VSFG imaging detects molecules only from the interface, the 4-wave method enables us to observe the signal from the bulk area. We concluded that the four repeating units of β-keratins in the bulk area which are suggested by X-ray diffraction studies are visualized in the 4-wave detected method. We also applied two IR super-resolution microscopies for the barb and discuss the site dependence of the orientation, distribution and concentration of β-keratin.