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This movie illustrates the principle used in photoactivated localization microscopy (PALM). A small fraction of a sample's fluorescent proteins are put into an "on" state, where they glow red when illuminated with yellow light. At the highest optical magnification each molecule looks like a fuzzy ball about 250 nm in diameter. However, the center where the fluorescent label is located can be determined to a fraction of that size. This is recorded by the smaller center spot. Next, a new sparse set of fluorescent proteins is turned on and the process iterates. In the top frame the accumulation of all the fuzzy balls forms the diffraction-limited image seen in a far-field microscope. The bottom frame shows the accumulation of the center spots, which builds a higher resolution PALM image.

Harald Hess