A microfluorimetric study of translational diffusion in erythrocyte membranes

A method is described which permits quantitative study of translational diffusion in the membranes of single cells. Human erythrocytes were labelled with fluorescein isothiocyanate and then hemolyzed, which yielded ghosts of normal shape and strong fluorescence. By application of sodium dodecylsulfatepolyacrylamide gel electrophoresis it was found that a very large part of fluorescein isothiocyanate was bound to the proteins of the erythrocyte membrane. In a fluorescence microscope, single ghosts were exposed to a sharply bounded intensive beam of light in such a manner that in each case only one half of the ghost was bleached. By microscopic measurements it was studied whether fluorescent material would diffuse from the unbleached part of the membrane into the bleached part and vice versa. However, within the measuring time of 20 min at room temperature a significant degree of such a diffusion could not be detected. In order to evaluate the experimental data quantitatively, the diffusion equation for a spherical surface was solved, and the obtained solution furthermore was integrated over the hemispheres. By these means a value of 3 · 10⁻¹² cm²/s was derived from the experimental data as an upper limit for the diffusion coefficient of fluorescein isothiocyanate-labelled compounds in the erythrocyte membrane at 20°–23°C.