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


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 


  1. 1.
    Hell, S.W.: Far-field optical nanoscopy. Science 316, 1153–1158 (2007)CrossRefADSGoogle Scholar
  2. 2.
    Egner, A., Hell, S.W.: Fluorescence microscopy with super-resolved optical sections. Trends Cell Biol. 15, 207–215 (2005)CrossRefGoogle Scholar
  3. 3.
    Hell, S.W., Stelzer, E.H.K.: Fundamental improvement of resolution with a 4Pi-confocal fluorescence microscope using two-photon excitation. Opt. Commun. 93, 277–282 (1992)CrossRefADSGoogle Scholar
  4. 4.
    Gustafsson, M.G., Agard, D.A., Sedat, J.W.: Sevenfold improvement of axial resolution in 3D widefield microscopy using two objective lenses. Proc. SPIE 2412, 147–156 (1995)CrossRefADSGoogle Scholar
  5. 5.
    Gustafsson, M.G., Agard, D.A., Sedat, J.W.: I5M: 3D widefield light microscopy with better than 100 nm axial resolution. J. Microsc. 195, 10–16 (1999)CrossRefGoogle Scholar
  6. 6.
    Heintzmann, R., Jovin, T.M., Cremer, C.: Saturated patterned excitation microscopy—a concept for optical resolution improvement. J. Opt. Soc. Am. A Opt. Image Sci. Vis. 19, 1599–1609 (2002)CrossRefADSGoogle Scholar
  7. 7.
    Gustafsson, M.G.: Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution. Proc. Natl. Acad. Sci. U.S.A. 102, 13081–13086 (2005)CrossRefADSGoogle Scholar
  8. 8.
    Hell, S.W., Wichmann, J.: Breaking the diffraction resolution limit by stimulated emission: stimulated emission depletion microscopy. Opt. Lett. 19, 780–782 (1994)ADSCrossRefGoogle Scholar
  9. 9.
    Hofmann, M., Eggeling, C., Jakobs, S., Hell, S.W.: Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins. Proc. Natl. Acad. Sci. U.S.A. 102, 17565–17569 (2005)CrossRefADSGoogle Scholar
  10. 10.
    Betzig, E., Patterson, G.H., Sougrat, R., Lindwasser, O.W., Olenych, S., Bonifacino, J.S., Davidson, M.W., Lippincott-Schwartz, J., Hess, H.F.: Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 (2006)CrossRefADSGoogle Scholar
  11. 11.
    Hess, S.T., Girirajan, T.P., Mason, M.D.: Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys. J. 91, 4258–4272 (2006)CrossRefADSGoogle Scholar
  12. 12.
    Rust, M.J., Bates, M., Zhuang, X.: Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793–795 (2006)CrossRefGoogle Scholar
  13. 13.
    Egner, A., Geisler, C., von Middendorff, C., Bock, H., Wenzel, D., Medda, R., Andresen, M., Stiel, A.C., Jakobs, S., Eggeling, C., Schonle, A., Hell, S.W.: Fluorescence nanoscopy in whole cells by asynchronous localization of photoswitching emitters. Biophys. J. 93, 3285–3290 (2007)CrossRefADSGoogle Scholar
  14. 14.
    Egner, A., Hell, S.W.: Fluorescence microscopy with super-resolved optical sections. Trends Cell Biol. 15, 207–215 (2005)CrossRefGoogle Scholar
  15. 15.
    Gugel, H., Bewersdorf, J., Jakobs, S., Engelhardt, J., Storz, R., Hell, S.W.: Cooperative 4Pi excitation and detection yields sevenfold sharper optical sections in live-cell microscopy. Biophys. J. 87, 4146–4152 (2004)CrossRefGoogle Scholar
  16. 16.
    Kano, H., Jakobs, S., Nagorni, M., Hell, S.W.: Dual-color 4Pi-confocal microscopy with 3D-resolution in the 100 nm range. Ultramicroscopy 90, 207–213 (2001)CrossRefGoogle Scholar
  17. 17.
    Nagorni, M., Hell, S.W.: 4Pi-confocal microscopy provides three-dimensional images of the microtubule network with 100- to 150-nm resolution. J. Struct. Biol. 123, 236–247 (1998)CrossRefGoogle Scholar
  18. 18.
    Shaner, N.C., Patterson, G.H., Davidson, M.W.: Advances in fluorescent protein technology. J. Cell Sci. 120, 4247–4260 (2007)CrossRefGoogle Scholar
  19. 19.
    Wiedenmann, J., Ivanchenko, S., Oswald, F., Nienhaus, G.U.: Identification of GFP-like proteins in nonbioluminescent, azooxanthellate anthozoa opens new perspectives for bioprospecting. Mar. Biotechnol. (NY) 6, 270–277 (2004)CrossRefGoogle Scholar
  20. 20.
    Wiedenmann, J., Vallone, B., Renzi, F., Nienhaus, K., Ivanchenko, S., Röcker, C., Nienhaus, G.U.: Red fluorescent protein eqFP611 and its genetically engineered dimeric variants. J. Biomed. Opt. 10, 14003 (2005)CrossRefGoogle Scholar
  21. 21.
    Nienhaus, K., Renzi, F., Vallone, B., Wiedenmann, J., Nienhaus, G.U.: Exploring chromophore–protein interactions in fluorescent protein cmFP512 from Cerianthus membranaceus: X-ray structure analysis and optical spectroscopy. Biochemistry 45, 12942–12953 (2006)CrossRefGoogle Scholar
  22. 22.
    Nienhaus, K., Renzi, F., Vallone, B., Wiedenmann, J., Nienhaus, G.U.: Chromophore-protein interactions in the anthozoan green fluorescent protein asFP499. Biophys. J. 91, 4210–4220 (2006)CrossRefADSGoogle Scholar
  23. 23.
    Ando, R., Hama, H., Yamamoto-Hino, M., Mizuno, H., Miyawaki, A.: An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein. Proc. Natl. Acad. Sci. U.S.A. 99, 12651–12656 (2002)CrossRefADSGoogle Scholar
  24. 24.
    Ando, R., Mizuno, H., Miyawaki, A.: Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting. Science 306, 1370–1373 (2004)CrossRefADSGoogle Scholar
  25. 25.
    Chudakov, D.M., Belousov, V.V., Zaraisky, A.G., Novoselov, V.V., Staroverov, D.B., Zorov, D.B., Lukyanov, S., Lukyanov, K.A.: Kindling fluorescent proteins for precise in vivo photolabeling. Nat. Biotechnol. 21, 191–194 (2003)CrossRefGoogle Scholar
  26. 26.
    Patterson, G.H., Lippincott-Schwartz, J.: A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297, 1873–1877 (2002)CrossRefADSGoogle Scholar
  27. 27.
    Wiedenmann, J., Ivanchenko, S., Oswald, F., Schmitt, F., Röcker, C., Salih, A., Spindler, K.D., Nienhaus, G.U.: EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion. Proc. Natl. Acad. Sci. U.S.A. 101, 15905–15910 (2004)CrossRefADSGoogle Scholar
  28. 28.
    Wiedenmann, J., Nienhaus, G.U.: Live-cell imaging with EosFP and other photoactivatable marker proteins of the GFP family. Expert Rev. Proteomics 3, 361–374 (2006)CrossRefGoogle Scholar
  29. 29.
    Nienhaus, G.U., Nienhaus, K., Hölzle, A., Ivanchenko, S., Renzi, F., Oswald, F., Wolff, M., Schmitt, F., Röcker, C., Vallone, B., Weidemann, W., Heilker, R., Nar, H., Wiedenmann, J.: Photoconvertible fluorescent protein EosFP: biophysical properties and cell biology applications. Photochem. Photobiol. 82, 351–358 (2006)CrossRefGoogle Scholar
  30. 30.
    Nienhaus, K., Nienhaus, G.U., Wiedenmann, J., Nar, H.: Structural basis for photo-induced protein cleavage and green-to-red conversion of fluorescent protein EosFP. Proc. Natl. Acad. Sci. U.S.A. 102, 9156–9159 (2005)CrossRefADSGoogle Scholar
  31. 31.
    Schneider, M., Barozzi, S., Testa, I., Faretta, M., Diaspro, A.: Two-photon activation and excitation properties of PA-GFP in the 720–920-nm region. Biophys. J. 89, 1346–1352 (2005)CrossRefGoogle Scholar
  32. 32.
    Tsutsui, H., Karasawa, S., Shimizu, H., Nukina, N., Miyawaki, A.: Semi-rational engineering of a coral fluorescent protein into an efficient highlighter. EMBO Rep. 6, 233–238 (2005)CrossRefGoogle Scholar
  33. 33.
    Ivanchenko, S., Glaschick, S., Röcker, C., Oswald, F., Wiedenmann, J., Nienhaus, G.U.: Two-photon excitation and photoconversion of EosFP in dual-color 4Pi confocal microscopy. Biophys. J. 92, 4451–4457 (2007)CrossRefADSGoogle Scholar
  34. 34.
    Ivanchenko, S., Röcker, C., Oswald, F., Wiedenmann, J., Nienhaus, G.U.: Targeted green-to-red photoconversion of EosFP, a fluorescent marker protein. J. Biol. Phys. 31, 249–259 (2005)CrossRefGoogle Scholar
  35. 35.
    Bestvater, F., Spiess, E., Stobrawa, G., Hacker, M., Feurer, T., Porwol, T., Berchner-Pfannschmidt, U., Wotzlaw, C., Acker, H.: Two-photon fluorescence absorption and emission spectra of dyes relevant for cell imaging. J. Microsc. 208, 108–115 (2002)CrossRefMathSciNetGoogle Scholar
  36. 36.
    Blab, G.A., Lommerse, O.H.M., Cognet, L., Harms, G.S., Schmidt, T.: Two-photon excitation action cross-sections of the autofluorescent proteins. Chem. Phys. Lett. 350, 71–77 (2001)CrossRefADSGoogle Scholar
  37. 37.
    Dickinson, M.E., Simbuerger, E., Zimmermann, B., Waters, C.W., Fraser, S.E.: Multiphoton excitation spectra in biological samples. J. Biomed. Opt. 8, 329–338 (2003)CrossRefADSGoogle Scholar
  38. 38.
    Xu, C., Webb, W.W.: Measurement of two-photon excitation cross sections of molecular fluorophores with data from 690 to 1050 nm. J. Opt. Soc. Am. B 13, 481–491 (1996)CrossRefADSGoogle Scholar
  39. 39.
    Kirkpatrick, S.M., Naik, R.R., Stone, M.O.: Nonlinear saturation and determination of the two-photon absorption cross section of green fluorescent protein. J. Phys. Chem. B. 105, 2867–2873 (2001)CrossRefGoogle Scholar
  40. 40.
    Schönle, A.: Imspector Image Acquisition and Analysis Software, v0.1. (2006)
  41. 41.
    Clendenon, J.L., Phillips, C.L., Sandoval, R.M., Fang, S., Dunn, K.W.: Voxx: a PC-based, near real-time volume rendering system for biological microscopy. Am. J. Physiol. Cell Physiol. 282, C213–218 (2002)Google Scholar
  42. 42.
    Baddeley, D., Carl, C., Cremer, C.: 4Pi microscopy deconvolution with a variable point-spread function. Appl. Opt. 45, 7056–7064 (2006)CrossRefADSGoogle Scholar
  43. 43.
    Lang, M.C., Engelhardt, J., Hell, S.W.: 4Pi microscopy with linear fluorescence excitation. Opt. Lett. 32, 259–261 (2007)CrossRefADSGoogle Scholar
  44. 44.
    Stracke, F., Weiss, B., Lehr, C.M., Konig, K., Schaefer, U.F., Schneider, M.: Multiphoton microscopy for the investigation of dermal penetration of nanoparticle-borne drugs. J. Invest. Dermatol. 126, 2224–2233 (2006)CrossRefGoogle Scholar
  45. 45.
    Gwinn, M.R., Vallyathan, V.: Nanoparticles: health effects—pros and cons. Environ. Health Perspect. 114, 1818–1825 (2006)Google Scholar

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

Personalised recommendations