Investigating mechanisms of collagen thermal denaturation by high resolution second-harmonic generation imaging

Abstract

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 degrees C to 70 degrees 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 degrees C does not produce any change in the averaged SHG intensity. At the heating temperature of 54 degrees C and above, we find that increasing the heating temperature and time leads to decreasing SHG intensity. As the tendon is heated above 54 degrees 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.

FIGURE 1

(A) Second-harmonic generation images of rat tail tendon treated at different heating temperatures and times. Structural change at the fibril level of the rat tail tendon is visible in the SHG signal as the fibril is heated at or above 54°C. Very little structural change is observed when the heating temperature is below 54°C. The images are 660 μm × 660 μm in size, and each one is a montage of 36 individually scanned images. (B) A large image of the untreated 50°C specimen from A, with a magnified view showing the resolved tendon fibrils.

FIGURE 2

SHG images, 660 μm × 660 μm, of rat tail tendon treated at temperatures below, just above, and far above the critical temperature of 54°C. At 40°C, the average intensity remains constant even for long heating time. At 55°C, just above the critical temperature, the SHG intensity from the tendon decreases with increasing heating time. At 60°C, 16°C above the critical temperature of 54°C, the SHG drops to near zero within few minutes of thermal treatment.

FIGURE 3

SHG intensity of rat tail tendon treated for different heating temperatures and times. For temperature above 54°C, the SHG intensity falls off rapidly with increasing heating time. Below 54°C, the intensities remain fairly constant with heating time. The relative intensities are normalized with respect to the SHG intensity measured at 57.5°C (0 min treatment).

FIGURE 4

(A) SHG image from a thermally treated rat tail tendon specimen where parts of image have little or no second-harmonic signal. The circled area shows an example of selected small area used to calculate the SHG intensity. (B) The SHG intensity average over five selected small areas for different heating temperatures and times. The SHG intensity falls off more slowly with increasing heating time than the case shown in Fig. 3.

FIGURE 5

Ratio of small to large area SHG intensity for different heating conditions plotted on logarithmic scale. For heating temperature above 54°C, the ratio rise with increasing heating time. The rise in the ratio with heating time suggests that the denaturation along the tiger-tail pattern occurs at a higher rate than the global melting of the collagen fibril.

FIGURE 6

Fibril counting procedure involves plotting the profile of the intensities along a selected line perpendicular to the fibril orientation. The line in the SHG image shows an example of such a selection. The profile plot corresponding to that selection is shown below the SHG image. The horizontal line in the profile plot is the average intensity along the selected line. It is used as a reference for counting the number of fibrils.

FIGURE 7

Number of fibril per unit length for different heating temperature and time. Five regions were selected for the counting. The count per unit length remains close to 0.62 counts per micron regardless of the heating temperatures and times.