Nonlinear imaging assisted ultrafast laser microsurgery for the treatment of vocal fold scarring

Femtosecond laser pulses achieve unrivaled microsurgical precision by developing extremely high peak intensity with relatively low total pulse energy. Despite a wide range of clinical advantages and applications that have been identified in bench-top studies, clinical development of femtosecond laser microsurgery outside of ophthalmology has remained in its infancy. The lack of a means to flexibly deliver the high-intensity laser light to areas of interest and guide it with suitable precision has constituted a serious hurdle to further clinical development. In response, this dissertation has detailed my research and development of table-top systems and the fiber-coupled femtosecond laser microsurgery scalpel to treat vocal fold scarring which does not have any reliable treatment in the clinic. This dissertation focuses on laser ablation and nonlinear imaging parameters for creation of sub-epithelial voids in vocal folds and how these parameters varied in scar tissue using animal models. We specifically investigated the differences in tissue architecture and scattering properties, and their relation to ablation thresholds and bubble lifetime. By using nonlinear imaging, we quantified tissue architecture and bubble dynamics. By developing a new method, we measured the ablation threshold below tissue surface while simultaneously extracting the extinction properties of different tissue layers. Also, we performed in-depth analysis using numerical, analytical, and experimental techniques to understand the limitation of maximum imaging depths with third-harmonic generation microscopy in turbid tissues such as vocal folds compared to two-photon autofluorescence microscopies. Our experimental results revealed that maximum imaging depth improved significantly from 140 µm to 420 µm using THG microscopy at 1552 nm excitation wavelength as compared to TPM at 776 nm. The second part of the dissertation explores developing a novel biomaterialdelivery method to inject and localize PEG 30 biomaterial inside sub-epithelial voids created by ultra-short laser pulses within scarred cheek pouch samples. To demonstrate the feasibility of this technique, we developed a semi-automated system to control and monitor the diffusion of the biomaterial inside scarred hamster cheek pouch samples. We observed a back-flow of the injected biomaterial along the point of injection and this condition prevented localization of the biomaterial at the desired locations without creating any void. In contrast to the biomaterial injection outcomes without any voids, the presence of sub-epithelial voids greatly reduced back-flow at the injection site and resulted in a lasting localization of the injected biomaterial at different locations of the tissue. We also performed a follow-up H&E histology and realized that the location and appearance of the biomaterial correlated well with TPAF and SHG in-situ nonlinear images. Finally, in the third part of the dissertation, we developed a piezo-scanned fiber device for high-speed ultrafast laser microsurgery, with an overall diameter of 5 mm. While the diameter of the scalpel is now half of our latest probe, its resolution has been also improved by 10% in both lateral and axial directions. The use of a high repetition rate fiber laser, delivering 300,000 pulses per second, and utilizing a sub-frame rate Lissajous scanning approach provided high ablation speeds suitable for clinical use. As shown by the uniform ablation of gold samples, an ablation FOV of 150 μm x 150 μm could be achieved within only 50 ms. With such ablation speeds, drilling into a cheek pouch tissue was possible using pulse energies of 200 nJ (3.2 J/cm2). With these speeds the surgeon could potentially move the surgery probe at speeds near 4 mm/s laterally in one direction while continuously removing a 150 μm wide tissue layer.