Fluorescence Ratio Imaging of Cytoplasmic pH in Plant Cells Using Fluorescein Diacetate
Spillard, Kristine S.
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Cytoplasmic pH is extremely important in the regulation of a variety of cellular processes. Microspectrofluorometry and fluorescence ratio imaging has been used to measure cytoplasmic pH in soybean ( Glycine max L. ) protoplasts and suspension cells, using the pH-sensitive probe fluorescein diacetate (FDA). The initially non-fluorescent derivative of fluorescein, FDA, is freely diffusible across the cell membrane into the cytoplasm where cellular esterases release the highly fluorescent chromophore. The fluorescein is retained in the cytoplasm and slowly diffuses out of the cells. pH measurements revealed a relatively acidic pH in normal protoplasts and cells, 6.4 + 0.2 and 6.5 ± 0.3 respectively, with a general increase in pHcyt upon treatment with weak base, and decrease with weak acid. Fluorescence ratio imaging of onion root epidermal cells shows higher cytoplasmic pH's from 6.0-7.0. In all cases ratio imaging revealed spatial variations of pH within the cytoplasm and pH differences between the cytoplasm and the nucleus were apparent.