Green fluorescent protein variants have been developed that report real-time change in pH and redox potential in living cells. The variants involve cysteine substitutions near the chromophore, which greatly alter the sensitivity of the protein to changes in its environment. Measurements can be made on single living cells in the fluorescence microscope or in cell suspension with an ordinary fluorimeter. The indicators are ratiometric by emission and/or excitation, which means that measurements at two different wavelengths are sufficient to determine both the quantity being measured and the indicator GFP concentration. The photophysics of a novel blue/green dual emission GFP variant will be presented.
The design principles, crystal structures and ultrafast spectroscopic analysis of probe response will be discussed in terms of atomic models involving excited state proton transfer. Some applications in living cells will be presented.