Optical stimulation of quantal exocytosis on transparent microchips

Photorelease of caged Ca is a uniquely powerful tool to study the dynamics of Catriggered exocytosis from individual cells. Using photolithography and other microfabrication techniques, we have developed transparent microchip devices to enable photorelease of caged Ca together with electrochemical detection of quantal catecholamine secretion from individual cells or cell arrays as a step towards developing high-throughput experimental devices. A 110 nm – thick transparent Indium-Tin-Oxide (ITO) film was sputter-deposited onto glass coverslips, which were then patterned into 24 cell-sized working electrodes (~20 μm by 20 μm). We loaded bovine chromaffin cells with acetoxymethyl (AM) ester derivatives of the Ca cage NP-EGTA and Ca indicator dye Fura-4F, then transferred these cells onto the working ITO electrodes for amperometric recordings. Upon flash photorelease of caged Ca, a uniform rise of [Ca]i within the target cell leads to quantal release of oxidizable catecholamines measured amperometrically by the underlying ITO electrode. We observed a burst of amperometric spikes upon rapid elevation of [Ca]i and a “priming” effect of substimulatory [Ca]i on the response of cells to subsequent [Ca]i elevation, similar to previous reports using different techniques. We conclude that UV photolysis of caged Ca is a suitable stimulation technique for higher-throughput studies of Ca-dependent exocytosis on transparent electrochemical microelectrode arrays.