Noninvasive Measurement of Bacterial Intracellular pH on a Single-Cell Level with Green Fluorescent Protein and Fluorescence Ratio Imaging Microscopy

We show that a pH-sensitive derivative of the green fluorescent protein, designated ratiometric GFP, can be used to measure intracellular pH (pH_i) in both gram-positive and gram-negative bacterial cells. In cells expressing ratiometric GFP, the excitation ratio (fluorescence intensity at 410 and 430 nm) is correlated to the pH_i, allowing fast and noninvasive determination of pH_i that is ideally suited for direct analysis of individual bacterial cells present in complex environments.