Phototoxicity is the most common problem investigators may encounter when performing live cell imaging. It develops due to excess laser exposure of cells loaded with fluorophores and can lead to often overlooked but significant artifacts, such as massive increase of intracellular Ca2+ concentration, which would make data interpretation problematic. Because information about laser- and dye-related changes in cytoplasmic calcium concentration is very limited, we aimed to describe this phenomenon to help investigators using laser scanning confocal microscopy in a non-invasive way. Therefore, in the present study we evaluated fluorescent fluctuations, which evolved in Fluo-3/4/8 loaded mouse pancreatic acinar cells during very low intensity laser excitation. We demonstrate that after standard loading procedure (2 μM Fluo-3/4/8-AM, 30 min at room temperature), applying 488 nm laser at as low as ca. 10 μW incident laser power (0.18 μW/μm2) at 1 Hz caused repetitive, 2-3 fold elevations of the resting intracellular fluorescence. The first latency and the pattern of the fluorescence fluctuations were laser power dependent and were related to Ca2+-release from intracellular stores, as they were abolished by BAPTA-AM treatment in Ca2+-free medium, but were not diminished by the reactive oxygen species (ROS) scavenger DMPO. Worryingly enough, the qualitative and quantitative features of the Ca2+-waves were practically indistinguishable from the responses evoked by secretagogue stimulation. Since using similar imaging conditions, a number of other cell types were reported to display spontaneous Ca2+ oscillations, we propose strategies to distinguish the real signals from artifacts.
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