Figure 2.

Calibration of pH-sensitive GFP variants. (A) A confocal image of a hippocampal CA3 pyramidal neuron expressing E²GFP (left). Calibration curve relating the fluorescence ratio of E²GFP expressing neurons to their intracellular pH (right; n = 4). E²GFP was used as an excitation ratiometric reporter, with excitation at two separate wavelengths (458 and 488 nm) and emitted light collected from a single window (500–550 nm). Intracellular pH was systematically varied by controlling extracellular pH in the presence of a proton-permeable ionophore (see Materials and Methods). Data was fit using established equations (Grynkiewicz et al., 1985; Arosio et al., 2010; see Materials and Methods) and pK_a was found to be 7.56. (B) A confocal image of a hippocampal CA3 pyramidal neuron expressing deGFP4 (left). Calibration curve relating the fluorescence ratio of deGFP4 expressing neurons to their intracellular pH (right; n = 7). deGFP4 was used as an emission ratiometric reporter, with excitation at a single wavelength (810 nm) and emission collected simultaneously at two separate windows (450–490 and 500–550 nm). The pH response properties of deGFP4 revealed a pK_a of 7.42. (C) A confocal image of a hippocampal CA1 pyramidal neuron expressing Cl-sensor (left). Calibration curve relating the fluorescence ratio of Cl-sensor expressing neurons to their intracellular pH (right; n = 7). Cl-sensor was used as an emission ratiometric reporter, with excitation at a single wavelength (850 nm) and emission collected simultaneously at two separate windows (460–500 and 520–550 nm). The pH response properties of Cl-sensor revealed a pK_a of 7.73.