In vivo and in vitro studies on fluorophore-specificity

Abstract

In vivo and in situ microscopy is a selective and easy method for detecting reactive oxygen (ROS)-and nitrogen species (RNS).Of the several fluorescent indicators developed in the last 30 years, the specificity and sensitivity of 4-amino-5-methylamino-2'-7'-difluorofluorescein diacetate (DAF-FM DA) as a nitric oxide (NO) indicator was tested by spectrofluorimetry and fluorescence microscopy.The peroxynitrite (ONOO -)-dependence of aminophenyl fluorescein (APF) and the hydrogen peroxide (H 2 O 2 )-sensitivity of 2'-7'-dichlorodihydrofluorescein diacetate (H 2 DCF DA) and 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red) was also determined.The results show that DAF-FM is a suitable fluorophore for detecting NO in plant tissues and aminophenyl fluorescein can be used as a ONOO --responsive dye.It was also found that DCF does not detect NO in solutions, but its fluorescence emission is strongly sensitive to H 2 O 2 .Moreover, the DCF fluorescence was found to be ONOO -sensitive, as well.In vivo studies revealed that Amplex Red can be applied as a H 2 O 2 -sensitive and -selective fluorophore in plant tissues.