Photobleaching recovery and anisotropy decay of green fluorescent protein GFP-S65T in solution and cells: Cytoplasmic viscosity probed by green fluorescent protein translational and rotational diffusion

The green fluorescent protein (GFP) was used as a noninvasive probe to quantify the rheological properties of. cell cytoplasm. GFP mutant S65T was purified from recombinant bacteria for solution studies, and expressed in CHO cell cytoplasm. GFP-S65T was brightly fluorescent in solution (lambda(ex) 492 nm, lambda(am) 509 nm) with a lifetime of 2.9 ns and a rotational correlation time (t(c)) of 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-mu m 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 O-2 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), was independent of spot diameter, and was unaffected by O-2 or quenchers. In cell cytoplasm, time-resolved microfluorimetry indicated a GFP lifetime of 2.6 ns and a t(c) of 36 +/- 3 ns, giving a relative viscosity (cytoplasm versus water) of 1.5. Photobleaching recovery of GFP in cytoplasm was 82 +/- 2% complete with a t(1/2) of 83 +/- 6 ms, giving a relative viscosity of 3.2. GFP translational diffusion increased 4.7-fold as cells swelled from a relative volume of 0.5 to 2. Taken together with measurements of GFP translation and rotation in aqueous dextran solutions, the data in cytoplasm support the view that the primary barrier to GFP diffusion is collisional interactions between GFP and macromolecular solutes.