OSA's Digital Library

Journal of the Optical Society of America A

Journal of the Optical Society of America A


  • Editor: Stephen A. Burns
  • Vol. 23, Iss. 5 — May. 1, 2006
  • pp: 1089–1095

Increasing the lateral resolution of 4Pi fluorescence microscopes

Nicolas Sandeau and Hugues Giovannini  »View Author Affiliations

JOSA A, Vol. 23, Issue 5, pp. 1089-1095 (2006)

View Full Text Article

Enhanced HTML    Acrobat PDF (584 KB)

Browse Journals / Lookup Meetings

Browse by Journal and Year


Lookup Conference Papers

Close Browse Journals / Lookup Meetings

Article Tools



The axial resolution of fluorescence microscopes can be considerably improved by superposing two illumination beams and by adding coherently the two wavefronts emitted by the luminescent sample. This solution has been implemented in 4Pi microscopes. Theoretical and experimental results have shown that a considerable improvement of the axial resolution can be obtained with these microscopes. However, the lateral resolution remains limited by diffraction. We propose a configuration of a 4Pi microscope in which the lateral displacement of the source modifies the collection efficiency function (CEF). Numerical calculations based on an approximate scalar theory and on exact vector-wave-optics results of the field distribution of the electromagnetic field in image space show that the lateral extent of the CEF can be reduced by a factor greater than 2 with respect to the diffraction limit. We show that, with this solution, the resolution in the transverse plane of 4Pi type B and 4Pi type C microscopes can be improved significantly.

© 2006 Optical Society of America

OCIS Codes
(180.1790) Microscopy : Confocal microscopy
(180.2520) Microscopy : Fluorescence microscopy
(260.3160) Physical optics : Interference
(300.2530) Spectroscopy : Fluorescence, laser-induced

ToC Category:

Original Manuscript: March 11, 2005
Revised Manuscript: August 30, 2005
Manuscript Accepted: November 6, 2005

Virtual Issues
Vol. 1, Iss. 6 Virtual Journal for Biomedical Optics

Nicolas Sandeau and Hugues Giovannini, "Increasing the lateral resolution of 4Pi fluorescence microscopes," J. Opt. Soc. Am. A 23, 1089-1095 (2006)

Sort:  Author  |  Year  |  Journal  |  Reset  


  1. M. Nagorni and S. W. Hell, "Coherent use of opposing lenses for axial resolution increase in fluorescence microscopy. I. Comparative study of concepts," J. Opt. Soc. Am. A 18, 36-48 (2001). [CrossRef]
  2. P.-F. Lenne, E. Etienne, and H. Rigneault, "Subwavelength patterns and high detection efficiency in fluorescence correlation spectroscopy using photonic structures," Appl. Phys. Lett. 80, 4106-4108 (2002). [CrossRef]
  3. S. Hell, "Double-confocal scanning microscope," European patent application EP0491289 (June 24, 1992).
  4. C. J. R. Sheppard and Y. Gong, "Improvement in axial resolution by interference confocal microscopy," Optik (Stuttgart) 87, 129-132 (1991).
  5. S. Hell and E. H. K. Stelzer, "Properties of a 4Pi confocal fluorescence microscope," J. Opt. Soc. Am. A 9, 2159-2166 (1992). [CrossRef]
  6. M. Gu and C. J. R. Sheppard, "Three-dimensional transfer functions in 4Pi confocal microscopes," J. Opt. Soc. Am. A 11, 1619-1627 (1994). [CrossRef]
  7. S. W. Hell, S. Lindek, C. Cremer, and E. H. K. Stelzer, "Measurement of the 4Pi-confocal point-spread function proves 75 nm axial resolution," Appl. Phys. Lett. 64, 1335-1337 (1994). [CrossRef]
  8. D. E. Koppel, D. Axelrod, J. Schlessinger, E. L. Elson, and W. W. Webb, "Dynamics of fluorescence marker concentration as a probe of mobility," Biophys. J. 16, 1315-1329 (1976). [CrossRef] [PubMed]
  9. C. M. Blanca, J. Bewersdorf, and S. W. Hell, "Single sharp spot in fluorescence microscopy of two opposing lenses," Appl. Phys. Lett. 79, 2321-2323 (2001). [CrossRef]
  10. M. Martinez-Corraz, A. Pons, and M. T. Caballero, "Axial apodization in 4Pi-confocal microscopy by annular binary filters," J. Opt. Soc. Am. A 19, 1532-1536 (2002). [CrossRef]
  11. M. Martinez-Corraz, M. T. Caballero, A. Pons, and P. Andrés, "Sidelobe decline in single-photon 4Pi microscopy by Toraldo rings," Micron 34, 319-325 (2003). [CrossRef]
  12. H. Gugel, J. Bewersdorf, S. Jakobs, J. Engelhardt, R. Storz, and S. W. Hell, "Cooperative 4Pi excitation and detection yields sevenfold sharper optical sections in live-cell microscopy," Biophys. J. 87, 4146-4152 (2004). [CrossRef] [PubMed]
  13. B. Richards and E. Wolf, "Electromagnetic diffraction in optical systems. II. Structure of the image field in an aplanatic system," Proc. R. Soc. London Ser. A 235, 358-379 (1959).
  14. J. Enderlein, "Theoretical study of detection of a dipole emitter through an objective with high numerical aperture," Opt. Lett. 25, 634-636 (2000). [CrossRef]
  15. M. Bohmer and J. Enderlein, "Orientation imaging of single molecules by wide-field epifluorescence microscopy," J. Opt. Soc. Am. B 20, 554-559 (2003). [CrossRef]
  16. S. H. Wiersma, P. Török, T. D. Visser, and P. Varga, "Comparison of different theories for focusing through a plane interface," J. Opt. Soc. Am. A 14, 1482-1490 (1997). [CrossRef]
  17. P. Torok, "Propagation of electromagnetic dipole waves through dielectric interfaces," Opt. Lett. 25, 1463-1465 (2000). [CrossRef]
  18. O. Haeberlé, "Focusing of light through a stratified medium: a practical approach for computing microscope point spread functions. Part I: Conventional microscopy," Opt. Commun. 216, 55-63 (2003). [CrossRef]
  19. M. Dyba and S. W. Hell, "Focal spots of size lambda/23 open up far-field fluorescence microscopy at 33 nm axial resolution," Phys. Rev. Lett. 88, 163901 (2002). [CrossRef] [PubMed]

Cited By

Alert me when this paper is cited

OSA is able to provide readers links to articles that cite this paper by participating in CrossRef's Cited-By Linking service. CrossRef includes content from more than 3000 publishers and societies. In addition to listing OSA journal articles that cite this paper, citing articles from other participating publishers will also be listed.

« Previous Article  |  Next Article »

OSA is a member of CrossRef.

CrossCheck Deposited