Quantitative X‐ray microanalysis of hydrogen peroxide within plant cells

Using quantitative X‐ray microanalysis in combination with CeCl 3 ‐based cytochemical staining of hydrogen peroxide (H 2 O 2 ) we have developed a new solution for quantification of H 2 O 2 at the subcellular level. Quantitative X‐ray microanalysis of plastic‐embedded leaves of Populus euphratica Oliv. showed that the obtained cerium precipitates by CeCl 3 staining were the mixture of cerium perhydroxides and cerium phosphate, in which the fractions of CePO 4 were: (1) 52–74% in cell walls of fresh leaf segments, and (2) 34–70% in the cytoplasm in 10 mM H 2 O 2 ‐treated leaf segments that were previously freeze‐dried. Taking the concentration of cerium phosphate as staining background, we reached the cellular concentration of cerium perhydroxides and the corresponding concentration of H 2 O 2 . Results showed that H 2 O 2 was present in the cytoplasm of rehydrated leaf segments (29–58 mM), but in fresh leaves, H 2 O 2 was observed in the walls of all measured cell types (17–74 mM). Microsc. Res. Tech., 2009. © 2008 Wiley‐Liss, Inc.