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Journal Article

4Pi-RESOLFT nanoscopy.

MPS-Authors
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Böhm,  U.
Department of NanoBiophotonics, MPI for biophysical chemistry, Max Planck Society;

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Hell,  S. W.
Department of NanoBiophotonics, MPI for biophysical chemistry, Max Planck Society;

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Schmidt,  R.
Department of NanoBiophotonics, MPI for biophysical chemistry, Max Planck Society;

Fulltext (public)

2247123.pdf
(Publisher version), 2MB

Supplementary Material (public)

2247123_Suppl_1.pdf
(Supplementary material), 3MB

2247123_Suppl_2.mpg
(Supplementary material), 8MB

2247123_Suppl_3.mpg
(Supplementary material), 8MB

2247123_Suppl_4.mpg
(Supplementary material), 8MB

Citation

Böhm, U., Hell, S. W., & Schmidt, R. (2016). 4Pi-RESOLFT nanoscopy. Nature Communications, 7: 10504. doi:10.1038/ncomms10504.


Cite as: http://hdl.handle.net/11858/00-001M-0000-0029-78FB-4
Abstract
By enlarging the aperture along the optic axis, the coherent utilization of opposing objective lenses (4Pi arrangement) has the potential to offer the sharpest and most light-efficient point-spread-functions in three-dimensional (3D) far-field fluorescence nanoscopy. However, to obtain unambiguous images, the signal has to be discriminated against contributions from lobes above and below the focal plane, which has tentatively limited 4Pi arrangements to imaging samples with controllable optical conditions. Here we apply the 4Pi scheme to RESOLFT nanoscopy using two-photon absorption for the on-switching of fluorescent proteins. We show that in this combination, the lobes are so low that low-light level, 3D nanoscale imaging of living cells becomes possible. Our method thus offers robust access to densely packed, axially extended cellular regions that have been notoriously difficult to super-resolve. Our approach also entails a fluorescence read-out scheme that translates molecular sensitivity to local off-switching rates into improved signal-to-noise ratio and resolution.