Characterization of cellular optoporation with distance

We have developed and characterized cellular optoporation with visible wave lengths of light using standard uncoated glass cover slips as the absorptiv e media. A frequency-doubled Nd:YAG laser pulse was focused at the interfac e of the glass surface and aqueous buffer, creating a stress wave and trans iently permeabilizing nearby cells. Following optoporation of adherent cell s, three spatial zones were present which were distinguished by the viabili ty of the cells and the loading efficiency (or number of extracellular mole cules loaded). The loading efficiency also depended on the concentration of the extracellular molecules and the molecular weight of the molecules. In the zone farthest from the laser beam (>60 mu m under these conditions), ne arly all cells were both successfully loaded and viable. To illustrate the wider applicability of this optoporation method, cells were loaded with a s ubstrate for protein kinase C and the cellular contents then analyzed by ca pillary electrophoresis. In contrast to peptides loaded by microinjection, optoporated peptide showed little proteolytic degradation, suggesting that the cells were minimally perturbed. Also demonstrating the potential for fu ture work, cells were optoporated and loaded with a fluorophore in the encl osed channels of microfluidic devices.