High-throughput microcavitation bubble induced cellular mechanotransduction

Focused pulsed laser irradiation allows for the deposition of energy with high spatial and temporal resolution. These attributes provide an optimal tool for non-contact manipulation in cellular biology such as laser microsurgery, cell membrane permeabilization, as well as targeted cell death.;In this thesis we investigate the direct physical effects produced by laser- generated microcavitation bubbles in adherent cell cultures. We examine how variation in pulse durations (180 ps - 6ns) and pulse energy (0.5 - 40 mu;J) affect microcavitation bubble (mu;CB) generated cell lysis, necrosis, and molecular delivery. To compare the effects of pulse duration we employ classical fluid dynamics modeling to quantify the perturbation caused on cell populations from mu;CB generated microTsunamis (a transient microscale burst of hydrodynamic shear stress). Through time-resolved imaging we capture the mu;CB dynamics at various energies and pulse durations. Moreover, the mathematical modeling provides information regarding the cellular exposure to time varying shear stress and impulse as a function of radial location from the mu;CB center. We demonstrate that the resultant cellular effect can be predicted based on the total impulse across a two order of magnitude span of pulse duration and pulse energy.;We also examine the region of cells beyond the zone of molecular delivery to investigate possible cellular reactions to mu;Tsunami exposure. Our studies have shown that cellular mechanotransduction occurs within cell populations spanning an area of up to 1 mm2 surrounding the mu;CB. Visualization of mechanotransduction is achieved through the visualization of intracellular calcium signaling via fluorescence microscopy that occurs due to the ability of the muTsunami generated shear stresses to stimulate G-protein coupled receptors at the apical cell surface. Moreover, we have shown that the observed signaling can be attenuated in a dose-dependent manner using 2-APB which is a known inhibitor to IP 3 induced Ca2+ release. This capability opens the development of a high-throughput screening platform for molecules that modulate cellular mechanotransduction. We have applied this approach to screen the effects of a small set of small molecules, in a 96-well plate in less than an hour. These detailed studies offer a basis for the design, development, and implementation of a novel high-throughput mechanotransduction assay to rapidly screen the effect of small molecules on cellular mechanotransduction at high throughput.