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Imaging techniques that allow intracellular pH and ion determination employ costly equipment and can have a long data acquisition time (minutes). We developed a novel fast principle to image the fluorescent properties of compound-specific dyes (Beutler et al. 2008, 2009). It is based on the ratio of emission upon light saturated excitation and precisely dosed low light excitation. The principle can cheaply be implemented in all confocal microscopes.
The value of the method was demonstrated by imaging internal pH in the cytoplasm and vacuoles of the sulfur oxidizing bacteria Beggiatoa sp. (Figure 1). This was considered as a method to evaluate predictions of genomic analyses, but appeared not possible with existing techniques. The RCSI method can be used for other compounds too, such as Ca2+ and Mg2+ concentration in living cells, while in sediments.
Figure 1: Transmission (gray) and pH (color-coded) images of a Beggiatoa filament. The vacuole is acidic (pH2), the cytoplasm is neutral (pH7-8). The ∆pH across the vacuole membrane was dissipated by nigericin, a Na+-H+ exchanger.