OSA's Digital Library

Virtual Journal for Biomedical Optics

Virtual Journal for Biomedical Optics


  • Editor: Gregory W. Faris
  • Vol. 4, Iss. 4 — Apr. 1, 2009

Limits for reduction of effective focal volume in multiple-beam light microscopy

Anton Arkhipov and Klaus Schulten  »View Author Affiliations

Optics Express, Vol. 17, Issue 4, pp. 2861-2870 (2009)

View Full Text Article

Enhanced HTML    Acrobat PDF (557 KB) Open Access

Browse Journals / Lookup Meetings

Browse by Journal and Year


Lookup Conference Papers

Close Browse Journals / Lookup Meetings

Article Tools



Employing interference patterns for illumination has been shown to reduce the focal volume in fluorescence microscopy. For example, the 4Pi technique employs two interfering laser beams and significantly decreases the focal volume, as compared to conventional microscopy. We study theoretically the effect of using multiple interfering laser beams on the focal volume. In realistic setups with three or four beams, the focal volume is about half of that from the 4Pi case. This improvement reaches a limit quickly as more beams are added, and for the idealized case of an infinite number of beams the focal volume is rather close to the three- or four-beam cases. Thus, our study suggests a limit for the possible reduction of the focal volume in a purely optical far-field setup.

© 2009 Optical Society of America

OCIS Codes
(110.0180) Imaging systems : Microscopy
(180.1790) Microscopy : Confocal microscopy
(180.2520) Microscopy : Fluorescence microscopy
(180.6900) Microscopy : Three-dimensional microscopy
(350.5730) Other areas of optics : Resolution

ToC Category:

Original Manuscript: December 22, 2008
Revised Manuscript: February 4, 2009
Manuscript Accepted: February 7, 2009
Published: February 11, 2009

Virtual Issues
Vol. 4, Iss. 4 Virtual Journal for Biomedical Optics

Anton Arkhipov and Klaus Schulten, "Limits for reduction of effective focal volume in multiple-beam light microscopy," Opt. Express 17, 2861-2870 (2009)

Sort:  Author  |  Year  |  Journal  |  Reset  


  1. S. Hell and E. H. K. Stelzer, "Fundamental improvement of resolution with a 4Pi-confocal fluorescence microscope using two-photon excitation," Opt. Commun. 93, 277-282 (1992). [CrossRef]
  2. B. Bailey, D. L. Farkas, D. L. Taylor, and F. Lanni, "Enhancement of axial resolution in fluorescence microscopy by standing-wave excitation," Nature 366, 44-48 (1993). [CrossRef] [PubMed]
  3. S. Lindek, R. Pick, and E. H. K. Stelzer, "Confocal Theta Microscope with Three Objective Lenses," Rev. Sci. Instrum. 65, 3367-3372 (1994). [CrossRef]
  4. M. G. L. Gustafsson, D. A. Agard, and J.W. Sedat, "Sevenfold improvement of axial resolution in 3D wide-field microscopy using two objective lenses," Proc. SPIE 2412, 147-156 (1995). [CrossRef]
  5. V. Krishnamurthi, B. Bailey, and F. Lanni, "Image processing in 3D standing-wave fluorescence microscopy," Proc. SPIE 2655, 18-25 (1996). [CrossRef]
  6. M. A. A. Neil, R. Juskaitis, and T. Wilson, "Method of obtaining optical sectioning by using structured light in a conventional microscope," Opt. Lett. 22, 1905-1907 (1997). [CrossRef]
  7. R. Heintzmann and C. G. Cremer, "Laterally modulated excitation microscopy: improvement of resolution by using a diffraction grating," Proc. SPIE 3568, 185-196 (1999). [CrossRef]
  8. M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, "I5M: 3D widefield light microscopy with better than 100 nm axial resolution," J. Microsc. 195, 10-16 (1999). [CrossRef] [PubMed]
  9. M. G. L. Gustafsson, "Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy," J. Microsc. 198, 82-87 (2000). [CrossRef] [PubMed]
  10. J. T. Frohn, H. F. Knapp, and A. Stemmer, "True optical resolution beyond the Rayleigh limit achieved by standing wave illumination," Proc. Natl. Acad. Sci. USA 97, 7232-7235 (2000). [CrossRef] [PubMed]
  11. G. E. Cragg and P. T. C. So, "Lateral resolution enhancement with standing evanescent waves," Opt. Lett. 25, 46-48 (2000). [CrossRef]
  12. O. Haeberle, C. Xu, A. Dieterlen, and S. Jacquey, "Multiple-objective microscopy with three-dimensional resolution near 100 nm and a long working distance," Opt. Lett. 26, 1684-1686 (2001). [CrossRef]
  13. P. T. C. So, H.-S. Kwon, and C. Y. Dong, "Resolution enhancement in standing-wave total internal reflection microscopy: a point-spread-function engineering approach," J. Opt. Soc. Am. A 18, 2833-2845 (2001). [CrossRef]
  14. J. Ryu, S. S. Hong, B. K. P. Horn, D. M. Freeman, and M. S. Mermelstein, "Multibeam interferometric illumination as the primary source of resolution in optical microscopy," Appl. Phys. Lett. 88, 171,112 (2006). [CrossRef]
  15. J. Swoger, J. Huisken, and E. H. K. Stelzer, "Multiple imaging axis microscopy improves resolution for thicksample applications," Opt. Lett. 28, 1654-1656 (2003). [CrossRef] [PubMed]
  16. S. W. Hell, "Far-Field Optical Nanoscopy," Science 316, 1153-1158 (2007). [CrossRef] [PubMed]
  17. S. W. Hell and J. Wichmann, "Breaking the diffraction resolution limit by stimulated emission," Opt. Lett. 19, 780-782 (1994). [CrossRef] [PubMed]
  18. M. G. L. Gustafsson, "Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution," Proc. Natl. Acad. Sci. USA 102, 13,081-13,086 (2005). [CrossRef]
  19. S. T. Hess, T. P. K. Girirajan, and M. D. Mason, "Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization Microscopy," Biophys. J. 91, 4258-4272 (2006). [CrossRef] [PubMed]
  20. E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, "Imaging Intracellular Fluorescent Proteins at Nanometer Resolution," Science 313, 1642-1645 (2006). [CrossRef] [PubMed]
  21. M. J. Rust, M. Bates, and X. Zhuang, "Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM)," Nat. Methods 3, 793-796 (2006). [CrossRef] [PubMed]
  22. L. Kastrup, H. Blom, C. Eggeling, and S. W. Hell, "Fluorescence Fluctuation Spectroscopy in Subdiffraction Focal Volumes," Phys. Rev. Lett. 94, 178104 (2005). [CrossRef] [PubMed]
  23. A. Arkhipov, J. Hüve, M. Kahms, R. Peters, and K. Schulten, "Continuous fluorescence microphotolysis and correlation spectroscopy using 4Pi microscopy," Biophys. J. 93, 4006-4017 (2007). [CrossRef] [PubMed]
  24. P. Schwille and E. Haustein, "Fluorescence Correlation Spectroscopy: A Tutorial for the Biophysics Textbook Online (BTOL)" (2002), http://www.biophysics.org/education/techniques.htm.
  25. M. C. Lang, T. Staudt, J. Engelhardt, and S. W. Hell, "4Pi microscopy with negligible sidelobes," New J. Phys. 10, 043041 (2008). [CrossRef]
  26. E. Wolf, "Electromagnetic diffraction in optical systems I. An integral representation of the image field," Proc. R. Soc. Lond. A. (Math. Phys. Sci.) 253, 349-357 (1959). [CrossRef]
  27. B. Richards and E. Wolf, "Electromagnetic diffraction in optical systems II. Structure of the image field in an aplanatic system," Proc. R. Soc. Lond. A. (Math. Phys. Sci.) 253, 358-379 (1959). [CrossRef]
  28. W. Humphrey, A. Dalke, and K. Schulten, "VMD - Visual Molecular Dynamics," J. Mol. Graphics 14, 33-38 (1996). [CrossRef]

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.


Fig. 1. Fig. 2. Fig. 3.
Fig. 4.

« Previous Article  |  Next Article »

OSA is a member of CrossRef.

CrossCheck Deposited