Live cell imaging using confocal microscopy induces intracellular calcium transients and cell death

Abstract

Isolated chondrocytes stained with fluo 4-AM and visualized using standard confocal microscopy techniques exhibited Ca2+ transients and oscillations. Decreasing the power of the laser light decreased the percentage of cells exhibiting these Ca2+ signals. Treatment with the antioxidant ascorbate reduced the Ca2+ response, suggesting that it was mediated by light-induced release of reactive oxygen species (ROS). Cell viability 24 h after the 1-h confocal imaging period was ∼90% for cells that were neither fluorescently stained nor subjected to laser excitation. By contrast, fluorescently stained cells imaged for 1 h exhibited greatly reduced viability. Treatment with ascorbate reduced the level of cell death, suggesting that the effect was mediated by release of exogenous ROS associated with the interaction of light and the fluorochrome. Ca2+oscillations were not always associated with cell death, suggesting that separate light-sensitive pathways mediate the two processes. Light-activated Ca2+ signaling may trigger alterations in numerous cell processes and thereby represent an important and hitherto overlooked artifact in fluorescent microscopy of viable cells.

Live-cell microscopy – tips and tools

“…However, these lamps might not produce enough light for very dim fluorophores or rapid imaging. Oxygen-radical scavengers such as ascorbic acid have been reported to be good anti-oxidants for live-cell imaging (Knight et al, 2003). Nonetheless, in our hands, scavenger concentrations of 100 μM did not make a significant difference in photobleaching rates under constant illumination (Fig.…”

Somatostatin Receptor Type 5 Modulates Somatostatin Receptor Type 2 Regulation of Adrenocorticotropin Secretion

“…The resting BSCOs pattern did not change during 50-min scanning except for a moderate increase in amplitude, which is reflected in the aNOA curve. The reason for this observation is not clear, and may be explained by the prolonged laser exposure of the cells as previously suggested in other cell systems (53). The moderate increase in aNOA actually further emphasizes the inhibitory results obtained with the ligands, with every inhibition or stimulation likely being stronger than the actual aNOA observed.…”

Chondrocyte Deformation Induces Mitochondrial Distortion and Heterogeneous Intracellular Strain Fields

“…This may occur through strain-mediated release of either nucleotides such as ATP (Graff et al 2000) or reactive oxygen species (ROS) (Ali et al 2004;Szafranski et al 2004). Indeed, articular chondrocytes have been shown to activate intracellular calcium signalling in response to both extracellular ATP (Elfervig et al 2001) and ROS (Knight et al 2003). Similar increased calcium signalling has also been reported for isolated chondrocytes compressed in agarose constructs, although the precise mechanisms are as yet unclear (Roberts et al 2001).…”