Journal of Biological Physics

, 33:433 | Cite as

Axial Resolution Enhancement by 4Pi Confocal Fluorescence Microscopy with Two-Photon Excitation

  • Sylvia Glaschick
  • Carlheinz Röcker
  • Karen Deuschle
  • Jörg Wiedenmann
  • Franz Oswald
  • Volker Mailänder
  • G. Ulrich Nienhaus
Original Paper

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.

Keywords

4Pi microscopy Axial superresolution Two-photon excitation Fluorescent proteins Photoactivatable proteins Nanoparticle–cell interaction 

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Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • Sylvia Glaschick
    • 1
  • Carlheinz Röcker
    • 1
  • Karen Deuschle
    • 1
  • Jörg Wiedenmann
    • 2
  • Franz Oswald
    • 3
  • Volker Mailänder
    • 4
  • G. Ulrich Nienhaus
    • 1
    • 5
  1. 1.Institute of BiophysicsUniversity of UlmUlmGermany
  2. 2.National Oceanography CentreUniversity of SouthamptonSouthamptonUK
  3. 3.Department of Internal Medicine IUniversity of UlmUlmGermany
  4. 4.Institute for Clinical Transfusion Medicine and Immunogenetics UlmUniversity of UlmUlmGermany
  5. 5.Department of PhysicsUniversity of Illinois at Urbana-ChampaignUrbanaUSA

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