Cell damage and reactive oxygen species production induced by fluorescence microscopy: effect on mitosis and guidelines for non‐invasive fluorescence microscopy
Top Cited Papers
- 15 September 2003
- journal article
- website
- Published by Wiley in The Plant Journal
- Vol. 36 (2), 280-290
- https://doi.org/10.1046/j.1365-313x.2003.01868.x
Abstract
The green fluorescent protein (GFP) and other intrinsically fluorescent proteins (IFPs) are popular reporters because they allow visualization of cellular constituents in living specimens. IFP technology makes it possible to view dynamic processes in living cells, but extended observation, using fluorescence microscopy (both wide‐field and confocal), can result in significant light energy exposure. Therefore, it is possible that cells experience light‐induced damage that alters cell physiology and confounds observations. To understand the impact that extended viewing has on cells, we obtained quantitative information about the effect of light energy dose and observation conditions on tobacco BY‐2 cell physiology. Our results show a non‐linear relationship between the excitation light intensity and mitotic arrest, and the frequency of mitotic arrest is dependent on the presence of an IFP that absorbs the excitation light. Moreover, fluorescence microscopy induces the production of reactive oxygen species (ROS), as assayed using BY‐2 cells loaded with oxidation‐sensitive dyes, and the level of ROS production increases if the cells express an IFP that absorbs the excitation light energy. The dye oxidation follows sigmoidal kinetics and is reversible if the cells are exposed to low irradiation levels. In addition, the dye oxidation rate shows a non‐linear relationship to the excitation light intensity, and a good correlation exists between photobleaching, mitotic arrest, and dye oxidation. The data highlight the importance of ROS scavenging for normal mitotic progression, and provide a reference for judiciously choosing conditions that avoid photobleaching that can lead to ROS accumulation and physiological damage.Keywords
This publication has 34 references indexed in Scilit:
- The spectroscopy of water vapour: Experiment, theory and applicationsPhysical Chemistry Chemical Physics, 2002
- Dynamic Analyses of the Expression of the HISTONE::YFP Fusion Protein in Arabidopsis Show That Syncytial Endosperm Is Divided in Mitotic DomainsPlant Cell, 2001
- Redistribution of Golgi Stacks and Other Organelles during Mitosis and Cytokinesis in Plant Cells,Plant Physiology, 2000
- Three-Dimensional Imaging by Deconvolution MicroscopyMethods, 1999
- Specific checkpoints regulate plant cell cycle progression in response to oxidative stressThe Plant Journal, 1999
- Confocal fluorescence microscopy of plant cellsProtoplasma, 1998
- THE GREEN FLUORESCENT PROTEINAnnual Review of Biochemistry, 1998
- ASCORBATE AND GLUTATHIONE: Keeping Active Oxygen Under ControlAnnual Review of Plant Physiology and Plant Molecular Biology, 1998
- Review article. Glutathione homeostasis in plants: implications for environmental sensing and plant developmentJournal of Experimental Botany, 1998
- Digital imaging fluorescence microscopy: spatial heterogeneity of photobleaching rate constants in individual cells.The Journal of cell biology, 1985