Cell damage and reactive oxygen species production induced by fluorescence microscopy: effect on mitosis and guidelines for non-invasive fluorescence microscopy

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.     

Oxyradical-induced GFP damage and loss of fluorescence

Small amounts of highly reactive oxygen species (oxyradicals) are normal by-products of cellular metabolism. However, under certain conditions large amounts of oxyradicals are generated inside cells which may cause extensive cellular damage. Not surprisingly, a large number of disease states have been linked to oxidative stress, including cancer, diabetes, Parkinson's disease, Alzheimer's disease, and heart disease. Previously, we had shown that fluorescence spectroscopy could be used to study the pH-dependence of GFP denaturation with various agents. In this report, we show that GFP readily loses its auto-fluorescence upon exposure to oxyradicals as measured by fluorescence spectroscopy. We further show that oxyradical scavengers can prevent this loss of GFP fluorescence, thus oxyradical-induced loss of GFP fluorescence could be used to screen for antioxidants. We have evaluated various parameters which could affect the sensitivity of this GFP-based oxyradical scavenging assay, such as concentration H(2)O(2) used to produce oxyradicals, pH of the buffer, as well as UV intensity. Surprisingly we found that pH had a very dramatic effect on oxyradical-induced GFP damage. GFP was found to be most susceptible to oxyradical-induced damage at pH 6.5, and least susceptible at pH 8.5. This is the first demonstration that GFP loses its fluorescence upon exposure to oxyradicals. Furthermore, the data presented here suggest that GFP could be used to develop assays to screen for antioxidants or radical scavengers.     

Kinase activity-dependent nuclear export opposes stress-induced nuclear accumulation and retention of Hog1 mitogen-activated protein kinase in the budding yeast Saccharomyces cerevisiae

Budding yeast adjusts to increases in external osmolarity via a specific mitogen-activated protein kinase signal pathway, the high-osmolarity glycerol response (HOG) pathway. Studies with a functional Hog1-green fluorescent protein (GFP) fusion reveal that even under nonstress conditions the mitogen-activated protein kinase Hog1 cycles between cytoplasmic and nuclear compartments. The basal distribution of the protein seems independent of its activator, Pbs2, and independent of its phosphorylation status. Upon osmotic challenge, the Hog1-GFP fusion becomes rapidly concentrated in the nucleus from which it is reexported after return to an iso-osmotic environment or after adaptation to high osmolarity. The preconditions and kinetics of increased nuclear localization correlate with those found for the dual phosphorylation of Hog1-GFP. The duration of Hog1 nuclear residence is modulated by the presence of the general stress activators Msn2 and Msn4. Reexport of Hog1 to the cytoplasm does not require de novo protein synthesis but depends on Hog1 kinase activity. Thus, at least three different mechanisms contribute to the intracellular distribution pattern of Hog1: phosphorylation-dependent nuclear accumulation, retention by nuclear targets, and a kinase-induced export.