Here we present two complementary methods for accurate diffusion measurements in yeast cell membranes.
Fluorescence spreading after photobleaching analyzes the blurring of an initially sharp border between bleached
and unbleached parts of the membrane. Two-focus scanning fluorescence correlation spectroscopy requires only
a low concentration of labeled fluorophores and allows for very long measurement times due to correction for
instabilities necessary to probe the slow diffusion in yeast plasma membranes. We apply these techniques to
study the dynamics of different transmembrane proteins in the plasma membrane of the yeast <i>Saccharomyces
cerevisiae</i>. The differences in the diffusion coefficients support the idea of co-existing membrane microdomains
in the yeast plasma membrane.