2006 Volume 14 Pages 9-18
Photodamage occurs, to a greater or lesser extent, during fluorescence bioimaging, and various procedures are empirically used to reduce the extent of this damage. However, measuring photodamage is difficult. Nitric oxide (NO), a gaseous messenger, is a free radical, and can be induced by light-exposure. In the present study, NO production was determined by the treatment of cultured cardiomyocytes, hippocampal neurons, dorsal root ganglion cells and serous peritoneal mast cells with 4, 5-diaminofluorescein (DAF-2). During laser irradiation in confocal microscopy, the fluorescence intensity of DAF-2 was increased in many punctuate regions of the cytoplasm as well as the nuclei of the cells examined. After the increase in NO levels, the beating of cardiomyocytes and the exocytosis of mast cells ceased. A stronger laser power caused a more intense peak and a more rapid increase in NO production. NO production in mast cells under various conditions was observed, in an attempt to reduce the extent of photodamage. Intermittent irradiation had no effect on NO production. Anti-oxidants (bovine serum albumin, vitamin E and melatonin), inhibited the peak and delayed the time course for NO production at a weak laser power, but had no effect on the bleaching of Indo-1, a fluorescent calcium indicator. In all bioimaging experiments in which a fluorescence technique is involved, the possibility of photodamage should be considered, and monitoring NO production by DAF-2 continues to be a relatively straightforward method for detecting photodamage.
