Pulsed laser microbeams for cellular manipulation : applications in cell biology and microfluidics

Highly focused pulsed laser radiation (pulsed laser microbeams) provide the ability to deposit energy with high spatial precision and controllable cellular damage. As a result, pulsed laser microbeams have been explored as a fast, non-contact means for cellular manipulations such as cellular microsurgery, transient cell membrane permeabilization, and targeted cell lysis. In this dissertation we examine the mechanisms of highly focused laser microbeams of nanosecond and picosecond duration to achieve cell lysis, cell necrosis, and molecular delivery. We have developed a time-resolved imaging system to visualize these processes with nanosecond temporal resolution and use image analysis to measure the physical perturbation applied to the cells as a function of laser microbeam pulse energy and pulse duration. Fluorescence assays are used to assess the biological response (necrosis, molecular delivery, and biochemical pathway activity) to the laser microbeam irradiation, and a biophysical model is developed to establish connections between specific physical characteristics and the resulting cellular effect. Our studies reveal that pulsed laser microbeam processes are mediated by optical breakdown resulting in plasma formation, shock wave emission, and cavitation bubble formation, expansion, and collapse. Cavitation bubble expansion was found to be the primary mechanism responsible for cellular modification. Hydrodynamic analysis based on the measured time evolution of the cavitation bubble growth, combined with assessment of the cellular response, revealed that the maximum wall shear stress associated with the cavitation bubble expansion governs the location and spatial extent of cell lysis, cell necrosis, and molecular delivery. In addition, we demonstrate how the variation of laser microbeam pulse duration can allow modulation of the spatial extent of cellular modification in order to tailor the cellular perturbations and optimize specific applications. These detailed studies provide a basis for the informed selection of specific laser parameters (i.e. pulse duration and energy) to achieve a desired cellular outcome, with controllable degrees of collateral damage. We demonstrate three different applications in which the laser parameters are well matched for particular applications in cellular analytics, activation of biochemical pathways, and microfluidic