Photobleaching of Fluorescent Probe in Microfluorometry and Detection of Active Oxygen Species

Abstract

It is important to understand the cause of photobleaching and to know how to protect from the bleaching the fluorescent DNA probes stained cells from bleaching in microscopy. We examined the protective effect of sodium azide (NaN₃) from photobleaching on the micrometer and the detection of active oxygen species produced in the probe solution by photoirradiation using an electron spin resonance (ESR) with a trapping agent. It was found that the bleaching of several probes was protected 11-99% by adding 1 mM NaN₃ as a scavenger for singlet oxygen. The NaN₃-pro-tecting efficiency for photobleaching on the microscope corresponded to the typical ESR signal intensity with the spin trapping agent for singlet oxygen. The OH radical produced in the probe-photosensitization was also detected by the ESR measurement with the another trapping agent. From the results, it was found that there were two types (predominantly, singlet oxygen or radicals) of active oxygen species produced at the probe-photoreactions. It was considered that active oxygen species produced from these photoreactions oxidized the probes to decrease the fluorescence intensity of the DNA probes used in the microscopy, and that the NaN₃ protected the photobleaching considerably without selfquenching of fluorescence.