A method for imaging of low pH in live cells based on excited state saturation

Imaging techniques that allow intracellular pH determination in ranges below pH 3 employ costly equipment and can have a long data acquisition time (minutes). Here, we describe a new methodology based on excited state saturation employing the fluorophore fluorescein-iso-thio-cyanate for confocal microscopy allowing a fast data acquisition in live organisms. To develop the method a model description of the fluorophore's molecular states was developed that led to a ratio function dependant on the excited states' lifetime. Due to the lifetime dependence on the pH of dissolved fluorescein-iso-thio-cyanate this ratio function was useful for pH determination. The model was tested theoretically and the pH dependence of the ratio function was verified experimentally with an artificial dye-bead system. Finally, a simple measuring protocol was developed allowing the automatic determination of the ratio function in images of live cells under the confocal microscope. This procedure was applied successfully to vacuolated Beggiatoa filaments with different internal pH values, near neutral in the cytoplasm and acidic in the vacuoles.