<p>Wild-type (WT) <i>Salmonella</i> was electroporated with 6 μM I-switch and recovered in SOC for 1 h at 37°C, followed by three washes with PBS. Bacteria were then clamped at various pH values in K<sup>+</sup>-rich buffer containing 40 μM nigericin for 1 h at room temperature (RT) and imaged on an Applied Precision Delta Vision inverted wide-field fluorescence microscope. The fluorescence intensity of both donor and acceptor channels of 50 cells were measured for every pH value indicated in the figure. The ratio of D/A intensities were obtained and the normalized D/A ratios were plotted as a function of pH, which yielded a sigmoidal intracellular calibration curve (<b>A</b>). The intracellular curve is overlaid on the in vitro calibration curve. Error bars represent mean ± standard deviation (SD). <b>(B)</b> Representative Donor (D), FRET (A) and D/A ratio images of WT <i>Salmonella</i> clamped at various pH values. Scale bar, 3 μm, color scale, bottom, right. Pseudocolor images were generated by calculating the D/A ratio per pixel. Using ImageJ software, the pixels were then color-coded using 16 colors with blue (D/A = 0.1) to red (D/A = 1) to indicate the transition from acidic to neutral pH.</p
