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.
Collagen, the most abundant protein in mammals, is the main component of connective tissues. It is responsible for the tensile strength in ligaments and tendons, the elasticity for skin, and the transparency and structural support for the cornea. The most prevalent collagen is Type I collagen, which is found in bones, tendons, and scar tissues. The triple helix of Type I collagen molecules are composed of polypeptide chains, each contains the repeated G-X-Y sequence, where G represents glycine, and X and Y usually correspond to usually praline or hydroxyproline (1). They combine to form microfibrils few nanometers in diameter, which then combine to form collagen fibrils that are few hundreds nanometers in diameter. The fibrils further bundles to form collagen fibers that are a few to hundreds of microns in diameter.
Thermally induced conformational changes in collagen have been actively studied, not only because collagen is the most abundant protein of the extracellular matrices, but also due to its relation to the application of several medical procedures. Examples include the use of heating to change cornea curvature, and the use of laser heating to stabilize shoulder joint and to rejuvenate skin (2-4). The response of collagen to heating has been studied using different methods including differential scanning calorimetry (DSC), x-ray diffraction, NMR, and spectroscopy (1,5,6). The denaturation of collagen is complicated among other variables by its polymeric nature and cross-linking. Despite numerous efforts, the precise mechanism of collagen denaturation remains unknown (1).
Among the various methods that can be used to study collagen denaturation, second-harmonic generation (SHG) microscopy is unique in that it is a nonlinear optical technique that has potential to be applied to collagen studies under in vivo conditions (7-13). In short, second-harmonic generation (SHG) is a coherent process in which two photons at the fundamental frequency are converted into one photon at twice the fundamental frequency. Due to the nonlinearity of the process. SHG occurs only in structures that lack inversion symmetry, and it has been demonstrated that Type 1 collagen is efficient in generating second-harmonic signals (7-13).
In this study, we used high resolution SHG microscopy to investigate the thermal denaturation of collagen from rat tail tendon. In particular, we obtain high resolution SHG images of heated collagen fibrils. We attempt to characterize the change in the SHG image as the collagen fibril undergoes thermally induced structural changes.
EXPERIMENTAL PROCEDURE
Sample preparation
Fresh rat tails obtained from the National Taiwan University Hospital were frozen at -80°C until before the experiment. Previous studies have found that freezing and thawing rat tail tendons do not affect their SHG signals (8,10). On the day of the experiment, the rat tails were first allowed to thaw at room temperature before the individual tendon fascicles were removed and placed in phosphate buffer saline (PBS) solutions (product specifications 0.01 M phosphate buffer, 0.0027 M potassium chloride, and 0.137 M sodium chloride, pH 7.4, at 25°C). The fascicles were then placed inside a tube filled with PBS solution and heated for a specific time in a thermal hath that was maintained at a given temperature. The heating temperature and lime were chosen based on a previous study where the SHG signal was found to change significantly between 50°C and 60°C for a heating lime of 10 min (7). After heating, the fascicles were placed on top of a PBS welted tissue paper, which was then placed on top of the glass slide to keep the fascicles moist. Finally a glass coverslip was added and sealed to prevent the tissue moisture from evaporation.