In fluorescence microscopy, the excitation light is focused to a spot on the specimen by the objective lens, which then captures the photons emitted by any fluorescent molecule that the beam has raised from a ground state to an excited state. Because the excitation spot is blurred according to the point spread function, fluorescent molecules that are closer than about 200 nm will be imaged as a single blurred spot. One approach to increasing the resolution is to switch all the fluorescent molecules at the periphery of the blurry excitation spot back to their ground state, or to a state where they no longer fluoresce in the normal way, leaving only those at the very center to be recorded. This can be done in practice by adding a second, very bright laser beam that wraps around the excitation beam like a torus. The wavelength and intensity of this second beam are adjusted so as to switch the fluorescent molecules off everywhere except at the very center of the point spread function, a region that can be as small as 20 nm across (Figure 9–37). The fluorescent probes used must be in a special class that is photoswitchable: their emission can be reversibly switched on and off with lights of different wavelengths. As the specimen is scanned with this arrangement of lasers, fluorescent molecules are switched on and off, and the small point spread function at each location is recorded. The diffraction limit is breached because the technique ensures that similar but very closely spaced molecules are in one of two different states, either fluorescing or dark. This approach is called STED (stimulated emission depletion microscopy) and various microscopes using versions of the general method are now in wide use. Resolutions of 20 nm have been achieved in biological specimens, and even higher resolution attained with nonbiological specimens (see Figure 9–37).
If a single fluorescent molecule is imaged, it appears as a circular blurry disc, but if sufficient photons have contributed to this image, the precise mathematical center of the disclike image can be determined very accurately, often to within a few nanometers. But the problem with a specimen that contains a large number
Figure 9–37 Superresolution microscopy can be achieved by reducing the size of the point spread function. (a) the size of a normal focused beam of excitatory light. (B) an extremely strong superimposed laser beam, at a different wavelength and in the shape of a torus, depletes emitted fluorescence everywhere in the specimen except right in the center of the beam, reducing the effective width of the point spread function (C). as the specimen is scanned, this small point spread function can then build up a crisp image in a process called steD (stimulated emission depletion microscopy). (D) synaptic vesicles in live cultured neurons, fluorescently labeled and imaged by ordinary confocal microscopy, with a resolution of 260 nm. (e) the same vesicles imaged by steD, with a resolution of 60 nm, which allows single vesicles to be resolved. (F) Fluorescently labeled replication factories in the nucleus of a cultured cell, imaged by ordinary confocal microscopy. (g) the same replication factories imaged by steD. single, discrete replication sites can be resolved by steD that cannot be seen in the confocal image. (a, B, and C, from g. Donnert et al., Proc. Natl Acad. Sci. USA 103:11440–11445, 2006. With permission from national academy of sciences; D and e, from V. Westphal et al., Science 320:246– 249, 2008. With permission from aaas; F and g, from Z. Cseresnyes,