title: 4Pi-RESOLFT nanoscopy.
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.
authors.affiliation: Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, Göttingen 37077, Germany.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Böhm
authors.affiliation: Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, Göttingen 37077, Germany.
authors.forename: Stefan W
authors.initials: SW
authors.lastname: Hell
authors.affiliation: Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, Göttingen 37077, Germany.
authors.forename: Roman
authors.initials: R
authors.lastname: Schmidt
references.citation: Proc Natl Acad Sci U S A. 2009 Mar 3;106(9):3125-30
references.pmid: 19202073
references.citation: Science. 1990 Apr 6;248(4951):73-6
references.pmid: 2321027
references.citation: Biochem J. 2007 Feb 15;402(1):35-42
references.pmid: 17117927
references.citation: Nano Lett. 2009 Jun;9(6):2497-500
references.pmid: 19449834
references.citation: J Microsc. 2004 Oct;216(Pt 1):49-51
references.pmid: 15369482
references.citation: Nat Methods. 2011 Apr;8(4):327-33
references.pmid: 21317909
references.citation: Nat Methods. 2008 Jun;5(6):539-44
references.pmid: 18488034
references.citation: Biophys J. 2005 Aug;89(2):1346-52
references.pmid: 15908572
references.citation: J Biol Phys. 2007 Dec;33(5-6):433-43
references.pmid: 19669529
references.citation: Nano Lett. 2011 Sep 14;11(9):3970-3
references.pmid: 21786833
references.citation: Q Rev Biophys. 2015 May;48(2):178-243
references.pmid: 25998828
references.citation: Nature. 2010 Nov 25;468(7323):580-4
references.pmid: 21107430
references.citation: Nano Lett. 2015 Jan 14;15(1):103-6
references.pmid: 25423166
references.citation: Nano Lett. 2009 Jun;9(6):2508-10
references.pmid: 19459703
references.citation: Nature. 2011 Oct 13;478(7368):204-8
references.pmid: 21909116
references.citation: Opt Lett. 2007 Feb 1;32(3):259-61
references.pmid: 17215938
references.citation: J Microsc. 2000 Mar;197(Pt 3):274-84
references.pmid: 10692131
references.citation: J Microsc. 1997 Jul;187(Pt 1):1-7
references.pmid: 9263436
references.citation: Nat Biotechnol. 2003 Nov;21(11):1347-55
references.pmid: 14595362
references.citation: Neuron. 2012 Sep 20;75(6):992-1000
references.pmid: 22998868
references.citation: Elife. 2012;1:e00248
references.pmid: 23330067
references.citation: Biophys J. 2004 Dec;87(6):4146-52
references.pmid: 15377532
references.citation: Opt Express. 2009 Aug 17;17(17):14567-73
references.pmid: 19687936