Extracellular pH imaging of a plant leaf with a polyelectrolyte multilayered nanosheet

We have developed a sheet-like pH imaging sensor based on a flexible and physically adhesive polymer thin film (referred to as a “pH sensing nanosheet”). The pH sensing nanosheet was composed of two films: one is a pH-sensitive layer-by-layer (LbL) film constructed from fluorescein-conjugated poly(acrylic acid) and poly(allylamine hydrochloride) and the other is a pH-insensitive film made from Nile red-embedded poly(d,l-lactic acid). The pH sensing nanosheet enabled the ratiometric imaging of pH changes in a leaf (500 × 500 μm(2)), namely the apoplastic ion milieu responding to an external NaCl stress. It was successfully mapped out that the alkalization of the leaf apoplast spread from the leaf base to the tip at 20 min after the stimulation and the pH value increased up to approximately pH 6.3 from less than pH 4.5 within 60 min when a 100 mM NaCl aqueous solution was added. The pH sensing nanosheet should be useful for energy metabolic mapping in tissue biology.