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Toward fluorescence nanoscopy

Abstract

For more than a century, the resolution of focusing light microscopy has been limited by diffraction to 180 nm in the focal plane and to 500 nm along the optic axis. Recently, microscopes have been reported that provide three- to sevenfold improved axial resolution in live cells. Moreover, a family of concepts has emerged that overcomes the diffraction barrier altogether. Its first exponent, stimulated emission depletion microscopy, has so far displayed a resolution down to 28 nm. Relying on saturated optical transitions, these concepts are limited only by the attainable saturation level. As strong saturation should be feasible at low light intensities, nanoscale imaging with focused light may be closer than ever.

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Figure 1: Examples of 4Pi confocal microscopy.
Figure 2: Actin cytoskeleton at the rim of a HeLa cell.
Figure 3: Optical sections from the microtubular network of a human embryonic kidney cell labeled by immunofluorescence.
Figure 4: Quantifying resolution.
Figure 5: Diffraction-unlimited spatial resolution with a reversible, saturable optical transition: the principle.
Figure 6: Physical conditions, setup and typical focal spot for STED.

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Acknowledgements

I thank M. Dyba, A. Egner, S. Jakobs, J. Jethwa, L. Kastrup, J. Keller and A. Schönle for constructive reading. The work of this laboratory has been funded by the Max Planck Society, with further support by the German Ministry of Research and Education, the Deutsche Forschungsgemeinschaft, and the Volkswagen Foundation.

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Correspondence to Stefan W Hell.

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Hell, S. Toward fluorescence nanoscopy. Nat Biotechnol 21, 1347–1355 (2003). https://doi.org/10.1038/nbt895

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