U. schwarz and C.M. green, BMC Cell Biol. 10:88, 2009.) of adjacent fluorescent molecules, as we saw earlier, is that they each contribute blurry, overlapping point spread functions to the image, making the exact position of any one molecule impossible to resolve. Another way round this limitation is to arrange for only a very few, clearly separated molecules to actively fluoresce at any one moment. The exact position of each of these can then be computed, before subsequent sets of molecules are examined.
In practice, this can be achieved by using lasers to sequentially switch on a sparse subset of fluorescent molecules in a specimen containing photoactivatable or photoswitchable fluorescent labels. Labels are activated, for example, by illumination with nearultraviolet light, which modifies a small subset of molecules so that they fluoresce when exposed to an excitation beam at another wavelength. These are then imaged before bleaching quenches their fluorescence and a new subset is activated. Each molecule emits a few thousand photons in response to the excitation before switching off, and the switching process can be repeated hundreds or even thousands of times, allowing the exact coordinates of a very large set of single molecules to be determined. The full set can be combined and digitally displayed as an image in which the computed location of each individual molecule is exactly marked (Figure 9–38). This class of methods has been variously termed photoactivated localization microscopy (PALM) or stochastic optical reconstruction microscopy (STORM).
By switching the fluorophores off and on sequentially in different regions of the specimen as a function of time, all the superresolution imaging methods described above allow the resolution of molecules that are much closer together than the 200 nm diffraction limit. In STED, the locations of the molecules are determined by using optical methods to define exactly where their fluorescence will be on or off. In PALM and STORM, individual fluorescent molecules are switched on and off at random over a period of time, allowing their positions to be accurately determined. PALM and STORM techniques have depended on the 100 photons 1000 photons 10,000 photons successive cycles of activation and bleaching allow well-separated single fuorescent molecules to be detected the exact center of each fuorescent molecule is determined and its position added to the map (C)a super-resolution image of the fuorescent structure is built up as the positions of successive small 1 µmgroups of molecules are added to the map
Figure 9–38 Single fluorescent molecules can be located with great accuracy. (a) Determining the exact mathematical center of the blurred image of a single fluorescent molecule becomes more accurate the more photons contribute to the final image. the point spread function described in the text dictates that the size of the molecular image is about 200 nm across, but in very bright specimens, the position of its center can be pinpointed to within a nanometer. (B) in this imaginary specimen, sparse subsets of fluorescent molecules are individually switched on briefly and then bleached. the exact positions of all these well-spaced molecules can be gradually built up into an image at superresolution. (C) in this portion of a cell, the microtubules have been fluorescently labeled and imaged at the top in a tirF microscope (see Figure 9–32) and below, at superresolution,  in a paLM microscope. the diameter of the microtubules in the lower panel now resembles their true size, about 25 nm,  rather than the 250 nm in the blurred image at the top. (a, from a.L. Mcevoy et al., BMC Biol. 8:106, 2010; C, courtesy of Carl Zeiss Ltd.) development of novel fluorescent probes that exhibit the appropriate switching behavior. All these methods are now being extended to incorporate multicolor imaging, threedimensional imaging (Figure 9–39), and livecell imaging in real time. Ending the long reign of the diffraction limit has certainly reinvigorated light microscopy and its place in cell biology research.