We have observed secretory granules beneath the plasma membrane of chromaffin cells. Using evanescent-field excitation by epillumination, we have illuminated a thin layer of cytosol where cells adhere to glass coverslips. Up to 800 frames could be recorded at diffraction-limited resolution without appreciable photodynamic damage. We localized single granules with an uncertainty of ~30 nm and tracked their motion in three dimensions. Granules in resting cells wander randomly as if imprisoned in a cage that leaves ~70 nm space around a granule. The 'cage' itself moves only slowly (D = 2 x 10-12 cm2/c). Rarely do granules arrive at or depart from the plasma membrane of resting cells. Stimulation increases lateral motion only slightly. After the plasma membrane has been depleted of granules by exocytosis, fresh granules can be seen to approach it at an angle. The method will be useful for exploring the molecular steps preceding exocytosis at the level of single granules.
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