On the use of fluorescence probes for detecting reactive oxygen and nitrogen species associated with photodynamic therapy

Fluorescent probes are frequently employed for the detection of different reactive oxygen and nitrogen species formed during the irradiation of photosensitized cells and tissues. Investigators often interpret the results in terms of information provided with the different probes without examining specificity or determinants of fluorogenic reactions. We examine five fluorescent probes in a cell-free system: reduced 2',7'-dichlorofluorescein, dihydroethidine, dihydrorhodamine, 3'-(p aminophenyl) fluorescein (APF), and 4',5'-diaminofluorescein. Of these, only APF demonstrates a high degree of specificity for a single reactive species. There is a substantial influence of peroxidase activity on all fluorogenic interactions. The fluorescence of the photosensitizing agent also must be taken into account in evaluating results.