Skip to main content
SpringerLink
Log in

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

  • Original Paper
  • Published:
Journal of Biological Physics Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Hell, S.W.: Far-field optical nanoscopy. Science 316, 1153–1158 (2007)

    Article  ADS  Google Scholar 

  2. Egner, A., Hell, S.W.: Fluorescence microscopy with super-resolved optical sections. Trends Cell Biol. 15, 207–215 (2005)

    Article  Google Scholar 

  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)

    Article  ADS  Google Scholar 

  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)

    Article  ADS  Google Scholar 

  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)

    Article  Google Scholar 

  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)

    Article  ADS  Google Scholar 

  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)

    Article  ADS  Google Scholar 

  8. Hell, S.W., Wichmann, J.: Breaking the diffraction resolution limit by stimulated emission: stimulated emission depletion microscopy. Opt. Lett. 19, 780–782 (1994)

    Article  ADS  Google Scholar 

  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)

    Article  ADS  Google Scholar 

  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)

    Article  ADS  Google Scholar 

  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)

    Article  ADS  Google Scholar 

  12. Rust, M.J., Bates, M., Zhuang, X.: Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793–795 (2006)

    Article  Google Scholar 

  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)

    Article  ADS  Google Scholar 

  14. Egner, A., Hell, S.W.: Fluorescence microscopy with super-resolved optical sections. Trends Cell Biol. 15, 207–215 (2005)

    Article  Google Scholar 

  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)

    Article  Google Scholar 

  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)

    Article  Google Scholar 

  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)

    Article  Google Scholar 

  18. Shaner, N.C., Patterson, G.H., Davidson, M.W.: Advances in fluorescent protein technology. J. Cell Sci. 120, 4247–4260 (2007)

    Article  Google Scholar 

  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)

    Article  Google Scholar 

  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)

    Article  Google Scholar 

  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)

    Article  Google Scholar 

  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)

    Article  ADS  Google Scholar 

  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)

    Article  ADS  Google Scholar 

  24. Ando, R., Mizuno, H., Miyawaki, A.: Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting. Science 306, 1370–1373 (2004)

    Article  ADS  Google Scholar 

  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)

    Article  Google Scholar 

  26. Patterson, G.H., Lippincott-Schwartz, J.: A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297, 1873–1877 (2002)

    Article  ADS  Google Scholar 

  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)

    Article  ADS  Google Scholar 

  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)

    Article  Google Scholar 

  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)

    Article  Google Scholar 

  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)

    Article  ADS  Google Scholar 

  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)

    Article  Google Scholar 

  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)

    Article  Google Scholar 

  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)

    Article  ADS  Google Scholar 

  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)

    Article  Google Scholar 

  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)

    Article  MathSciNet  Google Scholar 

  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)

    Article  ADS  Google Scholar 

  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)

    Article  ADS  Google Scholar 

  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)

    Article  ADS  Google Scholar 

  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)

    Article  Google Scholar 

  40. Schönle, A.: Imspector Image Acquisition and Analysis Software, v0.1. http://www.imspector.de (2006)

  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. Baddeley, D., Carl, C., Cremer, C.: 4Pi microscopy deconvolution with a variable point-spread function. Appl. Opt. 45, 7056–7064 (2006)

    Article  ADS  Google Scholar 

  43. Lang, M.C., Engelhardt, J., Hell, S.W.: 4Pi microscopy with linear fluorescence excitation. Opt. Lett. 32, 259–261 (2007)

    Article  ADS  Google Scholar 

  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)

    Article  Google Scholar 

  45. Gwinn, M.R., Vallyathan, V.: Nanoparticles: health effects—pros and cons. Environ. Health Perspect. 114, 1818–1825 (2006)

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institute of Biophysics, University of Ulm, 89069, Ulm, Germany

    Sylvia Glaschick, Carlheinz Röcker, Karen Deuschle & G. Ulrich Nienhaus

  2. National Oceanography Centre, University of Southampton, Southampton, SO14 3ZH, UK

    Jörg Wiedenmann

  3. Department of Internal Medicine I, University of Ulm, 89070, Ulm, Germany

    Franz Oswald

  4. Institute for Clinical Transfusion Medicine and Immunogenetics Ulm, University of Ulm, Helmholtzstr 10, 89081, Ulm, Germany

    Volker Mailänder

  5. Department of Physics, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA

    G. Ulrich Nienhaus

Authors
  1. Sylvia Glaschick
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Carlheinz Röcker
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Karen Deuschle
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jörg Wiedenmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Franz Oswald
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Volker Mailänder
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. G. Ulrich Nienhaus
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to G. Ulrich Nienhaus.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Glaschick, S., Röcker, C., Deuschle, K. et al. Axial Resolution Enhancement by 4Pi Confocal Fluorescence Microscopy with Two-Photon Excitation. J Biol Phys 33, 433–443 (2007). https://doi.org/10.1007/s10867-008-9084-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10867-008-9084-1

Keywords

Search

Navigation