The SHG microscope used in this study has been described previously.8–10 In short, an 80 MHz, 100-fs laser pulse from a ti-sa oscillator (Tsunami, Spectral Physics, Mountain View, CA) tuned to 780 nm was used as the excitation source. The laser beam, directed by scanning mirrors, was expanded and reflected onto the back aperture of the focusing objective by a short pass dichroic mirror (700dcspxruv-2p, Chroma Technology, Rockingham, VT). The oil-immersion objective (, S Fluor, NA1.3, Nikon, Japan) was used for imaging the RTT and MS, while for the corneas the water-immersion objective was used (, S Flour, NA 0.8, Nikon, Japan). For RTT and MS, the SHG signal from the sample was collected in the backward direction by the focusing objective and further guided by a dichroic mirror (435DCXR, Chroma technology), whereas SHG from corneas was detected in forward geometry using collection lens (, Chroma). In both cases, the SHG signals were isolated by a narrow band pass filter (HQ390/22m-2p, Chroma technology), and detected by photomultiplier tubes (R7400P, Hamamatsu, Japan).