We compare the use of two-dimensional total internal reflection fluorescence microscopy with a rapid, simple-to-implement method for three-dimensional (3D) imaging using spinning-disk confocal microscopy suitable for reliable 3D tracking of clathrin-coated endocytic and endosomal carriers. These carriers contain about 20 EGFP (enhanced green fluorescent protein) equivalents of a chimeric fluorescent protein (either clathrin light chain or one of the clathrin adaptor subunits). Under tissue culture conditions, the clathrin-containing carriers correspond to a variable number of relatively sparse, diffraction-limited, fluorescent objects that can be identified with a spatial precision of ~30 nm or better and a temporal resolution of <1 s. The applicability of these approaches to mammalian cells in culture allows investigators detailed monitoring of the composition dynamics of the clathrin-containing carriers which can then be used to study in living cells the molecular mechanisms required for the formation and traffic of clathrin-coated pits and vesicles.
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