A rapid Ca++ signal is known to be the main trigger for exocytosis in excitable cells. However, its mode of action is unknown. Recently, it has become clear that the spatial distribution of a Ca++ stimulus is important for exocytosis. To investigate this question we have developed a novel instrument capable of imaging Ca++ gradients in patch clamped cells. We have equipped a standard fluorescence microscope with a CCD camera and an image processing station. This combination can generate a thin section view of the fluorescence of a single cell. We have equipped this microscope with a pulsed laser illumination system. The distribution of intracellular calcium can be obtained by exciting the Ca++ indicator dye (e.g., rhod-2) with a brief laser pulse [300 ns long at 525 nm ], then an image can be formed with the light emitted by the dye. by synchronizing the laser pulse with a depolarizing stimulus in a patch-clamped chromaffin cell loaded with the fluorescent Ca++ indicator rhod-2, we could easily obtain snapshots of the Ca++ distribution at known times after a stimulus.
Pulsed laser imaging of rapid Ca2+ gradients in excitable cells