| This thesis explores the novel applications of femtosecond laser pulses as a tool for manipulating complex biological systems. Chapter 1 begins with a brief introduction on our current understanding of femtosecond laser pulse interactions with biological material. Much of what we know about the interaction process is based on theoretical simulations and assumptions, but this nevertheless provides an important framework for understanding why femtosecond lasers have received widespread attention. In this chapter, the non-linear multiphoton absorption and cascade ionization processes are discussed, along with ionized electron densities, shockwave and cavitation bubble formation.;With an understanding of the laser-tissue interaction process, Chapter 2 presents results on cell isolation and membrane surgery of living mammalian cells. Using focused femtosecond laser pulses, focal adhesions joining fibroblast cells were successfully removed, providing a novel method for cell isolation. Fibroblast isolation was shown to occur without inducing morphological damage to the cells. Membrane surgery was also performed on living cells. Dissection cuts were made along both the short and long axes of the cell. After membrane dissection, the cell maintained its morphology without evidence of cell collapse or disassociation.;Chapter 3 discusses the use of femtosecond laser pulses to transiently permeabilize living mammalian cells for the introduction of exogenous foreign material. Individual cells were laser pulse permeabilized in the presence of a hyperosmotic carbohydrate solution. Material delivery was confirmed by measuring the volumetric response of cells permeabilized in varying osmolarities of sugar. The survival of permeabilized cells in increasing hyperosmotic solutions was assessed using a membrane integrity assay. The intracellular accumulation of the sugar was theoretically determined using diffusion analysis.;Further demonstrating the novelty of the femtosecond laser, laser pulse surgery of an aquatic embryo, the zebrafish (Danio rerio), was also performed, Chapter 4. Utilizing transient pores that were formed in the embryonic cells of the zebrafish, an exogenous fluorescent probe, FITC, Streptavidin-conjugated quantum dots or plasmid DNA encoding EGFP (sCMV-EGFP) was introduced into the developing embryonic cells. Both FITC and the conjugated quantum dots were found to disperse throughout the cells as cleavage divisions of the blastomeres occurred. Gene expression of the plasmid DNA was seen in embryos reared to 24 hrs in various cells of the zebrafish body.;To determine if the laser pulse induced any short- or long-term effects on development, laser pulse manipulated embryos were reared to 2 and 7 days post-fertilization and compared to control embryos at the same developmental stages, Chapter 5. Light microscopy and scanning electron microscopy were used to compare whole body mosaics of the developed larvae. Key developmental morphological structures that were examined included the zebrafish fins, the ear, the olfactory pit and the neuromast patterning.;Chapter 6 concludes the thesis by presenting a brief discussion of future prospects for the applications of femtosecond laser pulses. |
