Cell permeabilization and molecular transport by laser microirradiation

Highly focused pulsed laser microbeams can be used to precisely dissect, inactivate, or perturb cells and subcellular targets. Here we introduce a new technique which employs pulsed microbeams to transiently permeabilize the plasma cell membrane and affect the delivery of molecules from the extracellular environment into the cell. This optically produced cell permeabilization can be applied using non-specific or specific modalities. In the non-specific modality, which we term 'optoporation', the pulsed microbeam is focused onto the glass coverslip on which the cells are plated. The generation of mechanical transients in connection with irradiation of the glass achieves molecular delivery to a number of cells proximal to the irradiation site. In the specific modality, termed 'optoinjection', the microbeam is focused directly onto the plasma cell membrane and achieves molecular delivery into that cell alone. To quantify the irradiation geometry involved in these and other microbeam processes, as well as examine the possibility of certain non-linear effects, we have developed a system using photochromic polymer films to characterize microbeam propagation and its effects within microirradiated targets. These photochromic polymers confirm that the laser microbeam are indeed focused to submicron dimensions within the targets in our systems. In addition the behavior of such polymers at higher pulse energies and irradiances indicate that multiphoton absorption and/or plasma formation may mediate some laser microirradiation processes.