Live cell imaging using confocal microscopy induces intracellular calcium transients and cell death.
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 percent-age 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 approximately 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.
Knight
Martin M
MM
Interdisciplinary Research Centre, Biomedical Materials and Medical Engineering Division, Department of Engineering, Queen Mary University of London, United Kingdom. m.m.knight@qmul.ac.uk
Roberts
Susan R
SR
Lee
David A
DA
Bader
Dan L
DL
D000814:Aniline Compounds; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D016477:Artifacts; D001205:Ascorbic Acid / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000528:radiation effects; D002358:Cartilage, Articular / Q000166:cytology / Q000502:physiology*; D002417:Cattle; D002470:Cell Survival / Q000528:radiation effects; D019902:Chondrocytes / Q000502:physiology*; D017484:Dermatitis, Phototoxic; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007425:Intracellular Membranes / Q000378:metabolism*; D007834:Lasers; D018613:Microscopy, Confocal; D017382:Reactive Oxygen Species / Q000378:metabolism; D014966:Xanthenes
D000814:Aniline Compounds; D000975:Antioxidants; D005452:Fluoresceins; D005456:Fluorescent Dyes; D017382:Reactive Oxygen Species; D014966:Xanthenes; C085925:calcein AM; C059715:Fluo-3; D001205:Ascorbic Acid; D002118:Calcium