Recently, there is compelling evidence that hydrogen peroxide (H₂O₂) and nitric oxide (NO) function as signaling molecules in plants, mediating a range of responses including stomatal movement. Thus, the choice of sensitive methods for detection of endogenous H₂O₂ and NO in guard cells are very important for understanding the role of H₂O₂ and NO in guard cell signaling. In addition, besides stomatal closure caused by interfering guard cell signaling, it can also be caused by widespread, nonspecific damage to guard cells. To determine whether stomatal movement is caused by damage to guard cells, sensitive methods for detection of guard cell viability are often required.

The oxidatively sensitive fluorophore 2′,7′-dichlorofluorecin (H₂DCF) is commonly employed to measure changes in intracellular H₂O₂ level directly. The non-polar diacetate ester (H₂DCFDA) of H₂DCF enters the cell and is hydrolysed into the more polar, non-fluorescent compound H₂DCF, which, therefore, is trapped. Subsequent oxidation of H₂DCF by H₂O₂, catalysed by peroxidases, yields the highly fluorescent DCF. Similarly, the cell-permeable, NO-sensitive fluorescent probe 4,5-diaminofluorescein diacetate (DAF-2DA) is widely used for the direct detection of NO presence in both animal and plant cells. The non-polar DAF-2DA enters the cell and is hydrolyzed by cytosolic esterase into the more polar, non-fluorescent compound DAF-2, which in the presence of NO is converted to the highly fluorescent triazole derivative DAF-2T. The fluorescent indicator dyes fluorescein diacetate (FAD) and propidium iodide (PI) are widely used for detection of cell viability. FAD passes through cell membranes and is hydrolyzed by intracellular esterase to produce a polar compound that passes slowly through a living cell membrane but fast through a damaged or dead cell membrane, and thus accumulates inside the viable cells and exhibits green fluorescence when excited by blue light. In contrast, PI passes through damaged or dead cell membranes and intercalates with DNA and RNA to form a bright red fluorescent complex seen in the nuclei of dying or dead cells but not living cells. Based on the above analysis, the fluorescent indicator dyes H₂DCFDA, DAF-2DA, FAD and PI load readily into guard cells, and their optical properties make them amenable to analysis by confocal laser scanning microscopy.

This protocol describes how to combine confocal laser scanning microscopy with fluorescent indicator dyes H₂DCFDA, DAF-2DA, FAD and PI respectively for measurement of H₂O₂ and NO and viability of guard cell in leaves of Arabidopsis (Arabidopsis thaliana).