Spatial control of cellular measurements with the laser micropipet

Continued progress in understanding cellular physiology requires new strate gies for biochemical measurements in solitary cells, multiple cells, and su bcompartments of cells. Large spatial gradients in the concentrations of mo lecules and presumably the activities of enzymes can occur in cells. Conseq uently, there is a critical need for measurement techniques for mammalian c ells with control over the numbers or regions of cells interrogated. In the present work, we developed a strategy to rapidly load the cytoplasmic cont ents of either multiple cells or a subregion of a single cell into a capill ary. A single, focused pulse from a laser created a mechanical shock wave w hich disrupted a group of cells or a portion of a cell in the path of the s hock wave. Simultaneously, the cytoplasm was loaded into a capillary for el ectrophoretic separation. The size of the region of cellular disruption (an d therefore the volume of cytoplasm collected) was controlled by the amount of energy in the laser pulse. Higher energies could be used to sample grou ps of cells while much lower energies could be utilized to selectively samp le the tip of a neuronal process. The feasibility of performing measurement s on subcellular compartments was also demonstrated by targeting reporter m olecules to these compartments. A reporter localized to the nucleus was det ected on the electropherogram following laser-mediated disruption of the ce ll and the nucleus. Finally, we demonstrate that this method terminated cel lular reactions with sufficient rapidity that cellular membrane repair mech anisms were not activated during cytoplasmic collection. The combined abili ty to preselect a spatial region of a cell or cells and to rapidly load tha t region into a capillary will greatly enhance the utility of CE in the bio chemical analysis of cells.