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Analysis of reactive oxygen species in the guard cell of wheat stoma with confocal microscope

Recently, the laser‐scanning confocal microscope has become a routine technique and indispensable tool for cell biological studies. Previous studies indicated that reactive oxygen species (ROS) were generated in tobacco epidermal cells with confocal microscope. In the present studies, the probe 2′,7′‐dichlorof luorescein diacetate (H2DCF‐DA) was used to research the change of ROS in the guard cell of wheat stoma, and catalase (CAT) was used to demonstrate that ROS had been labeled. The laser‐scanning mode of confocal microscope was XYT, and the time interval between two sections was 1.6351 s. Sixty optical sections were acquired with the laser‐scanning confocal microscope, and CAT (60,000 U mg−1) was added after four optical sections were scanned. Furthermore, the region of interest (ROI) was circled and the fluorescence intensity of ROS was quantified with Leica Confocal Software. The quantitative data were exported and the trend chart was made with software Excell. The results indicated that ROS were produced intracellularly in stomatal guard cells, and the quantified fluorescence intensity of ROS was declined with CAT added. It is a good method to research the instantaneous change of ROS in plant cells with confocal microscope and fluorescence probe H2DCF‐DA. Microsc. Res. Tech., 2011. © 2010 Wiley‐Liss, Inc.