ABSTRACT
Di raction l imits t he re solution t hat c an b e ob tained w ith v isible l ight m icroscopy to ∼200 nm, and has l imited biological applications of l ight microscopy for more than a c entury. Recently, novel superresolution techniques have succeeded in breaking the di raction barrier. ese techniques utilize t he principles of observation volume con nement, modulated illumination, and single molecule localization. Fluorescence photoactivation localization microscopy (FPALM) is an example of such a super-resolution light m icroscopy te chnique, w hich c an i mage xed o r l iving b iological s amples w ith a demo nstrated lateral resolution of ∼20-40 nm (Hess e t a l. 2 006, 2 007, G ould e t a l. 2 008). F PALM i nvolves re peated cycles of photo activation, localization, a nd photobleaching of m any sparse subsets of photo activatable molecules. FPALM is based on the principle that the positions of single molecules can be localized with a precision better than the di raction-limited resolution. Final images are rendered by plotting the positions and intensities of localized molecules, yielding a map of the distribution of those molecules with an optimal resolution given by the localization precision. Here, we present a detailed description of FPALM along with examples of results obtained from imaging biological samples.
