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Investigating Mechanisms of Collagen Thermal Denaturation by High Resolution Second-Harmonic Generation Imaging

Yen Sun ^*, Wei-Liang Chen ^*, Sung-Jan Lin  , Shiou-Hwa Jee , Yang-Fang Chen ^*, Ling-Chih Lin ^*, Peter T. C. So  and Chen-Yuan Dong ^*

^* Department of Physics, National Taiwan University, Taipei, Taiwan; Department of Dermatology, National Taiwan University Hospital and College of Medicine, Taipei, Taiwan; Institute of Biomedical Engineering, College of Medicine and College of Engineering, National Taiwan University, Taipei, Taiwan; and Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts

Correspondence: Address reprint requests to Peter T. C. So, E-mail: ptso{at}mit.edu; or Chen-Yuan Dong, E-mail: cydong{at}phys.ntu.edu.tw.

We apply the technique of second-harmonic generation (SHG) microscopy to obtain large area submicron resolution image of Type I collagen from rat tail tendon as it is heated from 40°C to 70°C for 0–180 min. The change in the collagen structure as reflected in its SHG image is observed at length scales from submicron to hundreds of microns. We observed that heating the tendon below the temperature of 54°C does not produce any change in the averaged SHG intensity. At the heating temperature of 54°C and above, we find that increasing the heating temperature and time leads to decreasing SHG intensity. As the tendon is heated above 54°C, the regions where the SHG signal vanish and form a tiger-tail like pattern. In addition, a decrease in the SHG signal occurs uniformly throughout the tendon. By comparing the relative SHG intensities in small and large areas, we found that the denaturation process responsible for forming the tiger-tail like pattern occurs at a higher rate than the global denaturation process occurring throughout the tendon. We also measured the fibril spacing and found that it remains constant at 1.61 ± 0.04 micron for all heating temperature and times. The constant fibril density shows that the global denaturation process occurs at a length scale smaller than the size of the fibril. Our results show that second-harmonic generation microscopy is effective in monitoring the thermal damage to collagen and has potential applications in biomedicine.