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Axial resolution enhancement by 4Pi confocal fluorescence microscopy with two-photon excitation

Axial resolution enhancement by 4Pi confocal fluorescence microscopy with two-photon excitation
Axial resolution enhancement by 4Pi confocal fluorescence microscopy with two-photon excitation
Confocal fluorescence microscopy and two-photon microscopy have become important techniques for the three-dimensional imaging of intact cells. Their lateral resolution is about 200–300 nm for visible light, whereas their axial resolution is significantly worse. By superimposing the spherical wave fronts from two opposing objective lenses in a coherent fashion in 4Pi microscopy, the axial resolution is greatly improved to ?100 nm. In combination with specific tagging of proteins or other cellular structures, 4Pi microscopy enables a multitude of molecular interactions in cell biology to be studied. Here, we discuss the choice of appropriate fluorescent tags for dual-color 4Pi microscopy and present applications of this technique in cellular biophysics. We employ two-color fluorescence detection of actin and tubulin networks stained with fluorescent organic dyes; mitochondrial networks are imaged using the photoactivatable fluorescent protein EosFP. A further example concerns the interaction of nanoparticles with mammalian cells.
4Pi microscopy, Axial superresolution, Two-photon excitation, Fluorescent proteins, Photoactivatable proteins, Nanoparticle, cell interaction
0092-0606
433-443
Glaschick, Sylvia
71d7e4b4-dcd6-4952-b60d-134fc672210c
Röcker, Carlheinz
e5752164-3ef7-47fe-887a-c2b750ea2b48
Deuschle, Karen
c264f117-529e-43e7-9a4d-a53971f7fcb5
Wiedenmann, Jörg
ad445af2-680f-4927-90b3-589ac9d538f7
Oswald, Franz
a5b02f2d-8439-411b-b5ad-999629cee58f
Mailänder, Volker
8975f8d1-ee11-4b1b-b4f6-90f1008fa8c6
Nienhaus, G. Ulrich
64eb2ac6-4fa9-416c-a066-f096d79307cb
Glaschick, Sylvia
71d7e4b4-dcd6-4952-b60d-134fc672210c
Röcker, Carlheinz
e5752164-3ef7-47fe-887a-c2b750ea2b48
Deuschle, Karen
c264f117-529e-43e7-9a4d-a53971f7fcb5
Wiedenmann, Jörg
ad445af2-680f-4927-90b3-589ac9d538f7
Oswald, Franz
a5b02f2d-8439-411b-b5ad-999629cee58f
Mailänder, Volker
8975f8d1-ee11-4b1b-b4f6-90f1008fa8c6
Nienhaus, G. Ulrich
64eb2ac6-4fa9-416c-a066-f096d79307cb

Glaschick, Sylvia, Röcker, Carlheinz, Deuschle, Karen, Wiedenmann, Jörg, Oswald, Franz, Mailänder, Volker and Nienhaus, G. Ulrich (2008) Axial resolution enhancement by 4Pi confocal fluorescence microscopy with two-photon excitation. Journal of Biological Physics, 33 (5-6), 433-443. (doi:10.1007/s10867-008-9084-1).

Record type: Article

Abstract

Confocal fluorescence microscopy and two-photon microscopy have become important techniques for the three-dimensional imaging of intact cells. Their lateral resolution is about 200–300 nm for visible light, whereas their axial resolution is significantly worse. By superimposing the spherical wave fronts from two opposing objective lenses in a coherent fashion in 4Pi microscopy, the axial resolution is greatly improved to ?100 nm. In combination with specific tagging of proteins or other cellular structures, 4Pi microscopy enables a multitude of molecular interactions in cell biology to be studied. Here, we discuss the choice of appropriate fluorescent tags for dual-color 4Pi microscopy and present applications of this technique in cellular biophysics. We employ two-color fluorescence detection of actin and tubulin networks stained with fluorescent organic dyes; mitochondrial networks are imaged using the photoactivatable fluorescent protein EosFP. A further example concerns the interaction of nanoparticles with mammalian cells.

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More information

Submitted date: December 2007
Published date: 2008
Keywords: 4Pi microscopy, Axial superresolution, Two-photon excitation, Fluorescent proteins, Photoactivatable proteins, Nanoparticle, cell interaction

Identifiers

Local EPrints ID: 58198
URI: http://eprints.soton.ac.uk/id/eprint/58198
ISSN: 0092-0606
PURE UUID: 2207b519-2048-4791-a04e-40cc62e5e085
ORCID for Jörg Wiedenmann: ORCID iD orcid.org/0000-0003-2128-2943

Catalogue record

Date deposited: 12 Aug 2008
Last modified: 16 Mar 2024 03:53

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Contributors

Author: Sylvia Glaschick
Author: Carlheinz Röcker
Author: Karen Deuschle
Author: Franz Oswald
Author: Volker Mailänder
Author: G. Ulrich Nienhaus

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