FIGURE 2 | Cellular features imaged by super-resolution techniques.

Aa | Point spread function (PSF) of stimulated emission depletion (STED) microscopy. The focal spot of excitation light (bright red) is overlapped with a doughnut-shaped red-shifted light (dark red), which quenches excited molecules in the excitation spot periphery. This confines emission to a central spot. Scanning this central spot (called the zero) across the sample results in a subdiffraction image. Ab | Synaptic vesicle movement imaged with STED. Synaptic vesicles were immunolabelled in live neurons, and the movement of each vesicle was individually recorded; the sum of 1,000 individual movie frames (1–1,000) depicts the movement of various synaptic vesicles. Ba | Stochastic optical reconstruction microscopy (STORM). The fluorescence image is constructed from highly precise localization of single molecules. In each imaging cycle, all fluorescent molecules in the field of view are switched off by, for example, a strong red laser. Only a small percentage of them are then switched on (green light) such that their images do not overlap, and their emission is recorded (red light) and used to localize their positions (white crosses) with nanometre accuracy. Bb | Multicolour and three-dimensional (3D) STORM imaging. Conventional (left panel) and STORM (right panel) images of immunostained microtubules (green) and clathrin-coated pits (red) in the same region of a BSC-1 cell. A 3D STORM image of a clathrin-coated pit is inset. An xy cross-section and an xz cross-section of the pit are shown in perspective. Ca | Photoactivated localization microscopy (PALM). PALM follows the same principle as STORM. To perform two-colour PALM imaging, the orange emitters (Eos fluorescent protein) are sequentially activated (405 nm light), imaged (561 nm light), localized and bleached until a subdiffraction image can be constructed (steps 1–3). After bleaching the remaining Eos molecules (step 4), the many active green emitters (Dronpa fluorescent protein) are first deactivated with a strong 488-nm light (step 5). Then, the green emitters are activated, imaged and bleached (steps 6–8). Cb | Two-colour PALM images show the nanostructural organization of cytoskeletal actin (green) and the adhesion protein paxillin (red) in an HFF-1 cell. Actin bundles densely cluster around some (arrowheads) but not all (full arrows) paxillin adhesions. Images in part Aa modified, with permission, from Ref. 32 © (2007) The Biophysical Society. Images in part Ab modified, with permission, from Ref. 40 © (2008) American Association for the Advancement of Science. Part Ba modified, with permission, from Nature Methods Ref. 24 © (2006) Macmillan Publishers Ltd. All rights reserved. Images in part Bb modified, with permission, from Ref. 46 © (2007) American Association for the Advancement of Science. Inset image in part Bb modified, with permission, from Ref. 49 © (2008) American Association for the Advancement of Science. Parts Ca, Cb modified, with permission, from Ref. 48 © (2007) National Academy of Sciences.

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