4Pi-RESOLFT nanoscopy

Nature Communications
Ulrike BöhmRoman Schmidt

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.

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Citations

Mar 7, 2017·Chemical Reviews·Hans Blom, Jerker Widengren
Feb 16, 2017·Optics Letters·Peng Gao, G Ulrich Nienhaus
Jun 16, 2016·F1000Research·Eric A SheldenJonathan C R Jones
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Apr 28, 2016·FEBS Letters·Janina HanneHans-Georg Kräusslich
Jan 30, 2019·Scientific Reports·Tamás GajdosMiklós Erdélyi
Oct 16, 2018·Frontiers in Immunology·Victoria JunghansPeter Jönsson
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Aug 10, 2017·Optics Express·Xiang HaoMartin J Booth
May 16, 2019·Biomedical Optics Express·Piotr ZdankowskiJason R Swedlow
Jan 13, 2021·Nature Biotechnology·Andreas BodénIlaria Testa
Jul 16, 2021·Applied Optics·Dong-Ryoung Lee, Joerg Bewersdorf
Aug 5, 2021·Biophysical Journal·Ulrike Boehm, Catherine G Galbraith
Jul 12, 2021·Biophysical Journal·René SiegmundAlexander Egner
May 24, 2019·The Journal of Physical Chemistry. B·Andrew RubaWeidong Yang
Jul 11, 2020·Bioconjugate Chemistry·Zhihe LiuChangfeng Wu
Feb 23, 2018··Jacopo AntonelloEdward Allgeyer

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Methods Mentioned

BETA
fluorescence microscopy
fluorescence imaging

Software Mentioned

iPALM
isoSTED
STORM
STED
PALM
RESOLFT

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