Green Fluorescent Protein as a Non-Invasive Probe of Viscosity and pH in Cell Cytoplasm and Organelles

Extract

Green fluorescent protein (GFP) was evaluated as a non-invasive probe to quantify the rheological properties and pH of intracellular aqueous compartments. Spectroscopic studies were done on several bacterially-expressed and purified GFP mutants. GFP-S65T was brightly fluorescent in solution (ƛ_ex 492 nm, ƛ_em 509 nm) with lifetime 2.9 ns and rotational correlation time ( t_c ) ∼20 ns. Recovery of GFP fluorescence after photobleaching was complete with a half-time (t_1/2) in aqueous saline of 30 ± 2 ms (5 μm diameter spot) giving a diffusion coefficient of 8.7 x 10^-7 cm^2/s. The t_1/2 was proportional to solution viscosity and was dependent on spot diameter. In contrast to fluorescein, GFP photobleaching efficiency was not affected by solution O₂ content, triplet state quenchers, singlet oxygen scavengers, and general radical quenchers. In solutions of higher viscosity, an additional, rapid GFP recovery process was detected and ascribed to reversible photobleaching. The t_1/2 for reversible photobleaching was 1.5-5.5 ms (relative viscosity 5-250), independent of spot diameter, and not affected by O₂ or quenchers.