Investigating second harmonic generation in collagen tissues

Collagen is the most abundant structural protein in the human body. When it is excited by femtosecond near infrared laser, second harmonic generation (SHG) signal at half the wavelength of the excitation wave is excited. For imaging thick tissues, the SHG signal is collected in the backward direction. The objective of this work is to elaborate the origin of the backward SHG in collagen at the fibril level and investigate some of its optic characteristics. The optic characteristics investigated include the wavelength dependence of SHG intensity, which is useful to analyze SHG in collagen tissues. However, the current published results are inconsistent. We study the microscopy system factors affecting the wavelength dependence and calibrate them by measuring the wavelength dependence of SHG intensity in a BaB₂O₄ crystal. With the proper calibration, typical wavelength dependence SHG spectra from mouse tail and Achilles tendon are investigated. The backward-collected SHG signal includes the backward generated SHG, and the forward generated but backward scattered SHG. Those two sources of the total backward SHG have different properties due to the difference in phase mismatch in the forward and backward directions. Here a non-invasive method is developed to separate them by using pinholes. By varying the pinhole size in a confocal multiphoton microscopy, the proportion of the backward scattered SHG to the total backward SHG can be obtained. Our results indicate that backward scattered SHG may not be the major source of backward SHG in the mouse tail tendon, which means significant SHG is purely generated in the backward direction. A large phase mismatch exists in generating backward SHG. Nevertheless, significant backward generated SHG has been observed in collagen tissues. We hypothesize that the periodic lattice structure of fibrillar collagen can provide a virtual momentum to assist the backward phase matching. Here the backward SHG phase matching is investigated in theory, simulation, and experiments, which are consistent and support the hypothesis. The various properties investigated in this thesis can provide a better understanding about SHG in collagen tissues and lead to new applications of SHG microscopy in diagnosing collagen related diseases in the future.Applied Science, Faculty ofElectrical and Computer Engineering, Department ofGraduat