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4. PALM

¡ò Principle
What fitting a PSF is to localization, photo-activated localization microscopy (PALM) is to "resolution", not true optical resolution. The basic premise of both techniques is to fill the imaging area with many dark fluorophores that can be photoactivated into a fluorescing state by a flash of light. Because photoactivation is stochastic, only a few, well separated molecules "turn on." Then Gaussians are fit to their PSFs to high precision. After the few bright dots photobleach, another flash of the photoactivating light activates random fluorophores again and the PSFs are fit of these different well spaced objects. This process is repeated many times, building up an image molecule-by-molecule; and because the molecules were localized at different times, the "resolution" of the final image can be much higher than that limited by diffraction.

¡ò Advantage
? We can observe the molecule-level materials in three dimensions.