CYTOPLASMIC VISCOSITY NEAR THE CELL PLASMA-MEMBRANE - TRANSLATIONAL DIFFUSION OF A SMALL FLUORESCENT SOLUTE MEASURED BY TOTAL INTERNAL REFLECTION-FLUORESCENCE PHOTOBLEACHING RECOVERY

Total internal reflection-fluorescence recovery after photobleaching ( TIR-FRAP) was applied to measure solute translational diffusion in the aqueous phase of membrane-adjacent cytoplasm, TIR fluorescence excita tion in aqueous solutions and fluorescently labeled cells was produced by laser illumination at a subcritical angle utilizing a quartz prism ; microsecond-resolution FRAP was accomplished by acousto-optic modula tors and electronic photomultiplier gating, A mathematical model was d eveloped to determine solute diffusion coefficient from the time cours e of photobleaching recovery, bleach time, bleach intensity, and evane scent field penetration depth; the model included irreversible and rev ersible photobleaching processes, with triplet state diffusion. The va lidity and accuracy of TIR-FRAP measurements were first examined in aq ueous fluorophore solutions. Diffusion coefficients for fluorescein is othiocyanate-dextrans (10-2000 kDa) determined by TIR-FRAP (recovery t (1/2) 0.5-2.2 ms) agreed with values measured by conventional spot pho tobleaching. Model predictions for the dependence of recovery curve sh ape on solution viscosity, bleach time, and bleach depth were validate d experimentally using aqueous fluorescein solutions, To study solute diffusion in cytosol, MDCK epithelial cells were fluorescently labeled with the small solute boxyethyl-5-carboxyfluorescein-acetoxymethyl-es ter (BCECF), A reversible photobleaching process (t(1/2) approximate t o 0.5 ms) was identified that involved triplet-state relaxation and co uld be eliminated by triplet-state quenching with 100% oxygen, TIR-FRA P t(1/2) values for irreversible BCECF bleaching, representing BCECF t ranslational diffusion in the evanescent field, were in the range 2.2- 4.8 ms (0.2-1 ms bleach times), yielding a BCECF diffusion coefficient 6-10-fold less than that in water, These results establish the theory and the first experimental application of TIR-FRAP to measure aqueous -phase solute diffusion, and indicate slowed translational diffusion o f a small solute in membrane-adjacent cytosol.