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Spherical nanosized focal spot unravels the interior of cells

Nature Methods volume 5, pages 539544 (2008) | Download Citation

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Abstract

The resolution of any linear imaging system is given by its point spread function (PSF) that quantifies the blur of an object point in the image. The sharper the PSF, the better the resolution is. In standard fluorescence microscopy, however, diffraction dictates a PSF with a cigar-shaped main maximum, called the focal spot, which extends over at least half the wavelength of light (λ = 400–700 nm) in the focal plane and >λ along the optical axis (z). Although concepts have been developed to sharpen the focal spot both laterally and axially, none of them has reached their ultimate goal: a spherical spot that can be arbitrarily downscaled in size. Here we introduce a fluorescence microscope that creates nearly spherical focal spots of 40–45 nm (λ/16) in diameter. Fully relying on focused light, this lens-based fluorescence nanoscope unravels the interior of cells noninvasively, uniquely dissecting their sub-λ–sized organelles.

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Acknowledgements

We thank S. Stoldt for help with cell culture. We acknowledge A. Schönle and J. Keller (Max Planck Institute for Biophysical Chemistry) for providing us with the analysis software (Imspector), the PSF calculation software and for helpful discussions. We also acknowledge helpful discussions with K. Willig and thank J. Jethwa for critically reading the manuscript. This work was supported by grants of the Deutsche Forschungsgemeinschaft (JA 1129/3) to S.J. and (SFB 755) to A.E. and S.W.H.

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Affiliations

  1. Max Planck Institute for Biophysical Chemistry, Department of NanoBiophotonics, Am Fassberg 11, 37077 Göttingen, Germany.

    • Roman Schmidt
    • , Christian A Wurm
    • , Stefan Jakobs
    • , Alexander Egner
    •  & Stefan W Hell
  2. German Cancer Research Center (DKFZ), High-Resolution Optical Microscopy Division, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.

    • Johann Engelhardt
    •  & Stefan W Hell

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Correspondence to Alexander Egner or Stefan W Hell.

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DOI

https://doi.org/10.1038/nmeth.1214

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