Here we describe a technique that allows us to visualise, in real time the formation and dynamics (fusion, changes of shape and translocation) of vacuoles in living cells. The technique involves infusion of a dextran - bound fluorescent probe into the cytosol of the cell via a patch pipette, using the whole-cell patch clamp configuration. Experiments were conducted on pancreatic acinar cells stimulated with supramaximal concentrations of cholecystokinin (CCK). The vacuoles, forming in the cytoplasm of the cell, were revealed as dark imprints on a bright fluorescence background, produced by the probe and visualised using confocal microscopy. A combination of two dextran-bound probes - one infused into the cytosol and the second added to the extracellular solution was used to identify endocytic and non-endocytic vacuoles. The cytosolic dextran-bound probe was also used together with a Golgi indicator to illustrate the possibility of combining the probes and identifying the localisation of vacuoles with respect to other cellular organelles in pancreatic acinar cells. Combinations of cytosolic dextran-bound probes with ER or mitochondrial probes were also used to simultaneously visualise vacuoles and corresponding organelles. We expect that the new technique will also be applicable and useful for studies of vacuole dynamics in other cell types.