Lens Aberrations in Confocal Fluorescence Microscopy

  • Stefan W. Hell
  • Ernst H. K. Stelzer


Modern optical microscopes are so good that many scientists forget that these instruments only provide their optimal performance if they are used under certain operating conditions. Typical users may be unaware of the very existence of such limitations because they may unwittingly work within the limits or fail to recognize their effects. It is probably also correct to assume that the engineers who designed the instrument did not expect the scientist to use it with devices that exceed the sensitivity and intra-scene dynamic range of the human eye or photographic film. Last but not least, the manufacturer does not intend to discourage purchase by emphasizing the limits imposed by the specifications of the instrument.


Objective Lens Numerical Aperture Mount Medium Axial Resolution Focal Shift 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Born, M., and Wolf, E., 1980, Principles of Optics, Pergamon Press, Oxford.Google Scholar
  2. Carlsson, K., 1991, The influence of specimen refractive index, detector signal integration, and non-uniform scan speed on the imaging properties in confocal microscopy, J. Microsc. 163:167–178.CrossRefGoogle Scholar
  3. Denk, W., Strickler, J.H., and Webb, W.W., 1990, Two-photon laser scanning fluorescence microscopy, Science 248:73–76.PubMedCrossRefGoogle Scholar
  4. Hell, S., Reiner, G., Cremer, C., and Stelzer, E.H.K., 1993, Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index, J. Microsc. 169:391–405.CrossRefGoogle Scholar
  5. Hell, S., and Stelzer, E.H.K., 1992, Fundamental improvement of resolution with a 4Pi-confocal fluorescence microscope using two-photon excitation, Optic Commun. 93:277–282.CrossRefGoogle Scholar
  6. Hell, S., Lehtonen, E., and Stelzer, E.H.K., 1992, Confocal fluorescence microscopy: Wave optics considerations and applications to cell biology. In: Visualization in Biomedical Microscopies (A. Kriete, ed.), Verlag Chemie, Weinheim, pp. 145–161.Google Scholar
  7. Hopkins, H.H. 1943, The airydisc formula for systems of high relative aperture, Proc. Phys. Soc. 55:116.CrossRefGoogle Scholar
  8. Kaiser, W., and Garrett, C.G.B., 1961, Phys. Rev. Lett. 7:229.CrossRefGoogle Scholar
  9. Li, Y., and Wolf, E., 1981, Focal shifts in diffracted converging spherical waves, Optic Commun. 39:211–215.CrossRefGoogle Scholar
  10. Ling, H., and Lee, S.W., 1984, Focusing of electromagnetic waves through a dielectric interface, J. Opt. Soc. Am. A1:965–973.CrossRefGoogle Scholar
  11. Richards, B., and Wolf, E., 1959, Electromagnetic diffraction in optical systems. II, Proc. R. Soc. Lond. Ser. A 253:349–357.CrossRefGoogle Scholar
  12. Shaw, P.J., and Rawlins, D.J., 1991, The point-spread function of a confocal microscope: Its measurement and use in deconvolution of 3-D data, J. Microsc. 163:151–166.CrossRefGoogle Scholar
  13. Sheppard, C.J.R., 1988, Aberrations in high aperture conventional and confocal imaging systems, Appl. Optics 27:4782–4786.CrossRefGoogle Scholar
  14. Sheppard, C.J.R., and Cogswell, C.J., 1991, Effects of aberrating layers and tube length on confocal imaging properties, Optik 87:34–38.Google Scholar
  15. Sheppard, C.J.R., and Gu, M., 1992, Image formation in two-photon fluorescence microscopy, Optik 86:104–106 [changed in an Erratum, Optik 92:102:1992].Google Scholar
  16. Stelzer, E.H.K., Hell, S., Lindek, S., Stricker, R., Pick, R., Storz, C., Ritter, G., and Salmon, N., 1994, Nonlinear absorption extends confocal fluorescence microscopy into the ultra-violet regime and confines the illumination volume, Optic Commun. 104:223–228.CrossRefGoogle Scholar
  17. Stelzer, E.H.K., Wacker, I., and De Mey, J.R., 1991, Confocal fluorescence miocroscopy in modern cell biology, Semin. Cell Biol. 2:145–152.PubMedGoogle Scholar
  18. Van-der-Voort, H.T.M., and Brakenhoff, G.J., 1990, 3-D image formation in high-aperture fluorescence confocal microscopy: A numerical analysis, J.Microsc. 158:43–54.CrossRefGoogle Scholar
  19. Visser, T.D., Oud, J.L., and Brakenhoff, G.J., 1992, Refractive index and axial distance measurements in 3-D microscopy, Optik 90:17–19.Google Scholar
  20. Visser, T.D., Brakenhoff, G.J., and Groen, F.C.A., 1991, The one-point fluorescence response in confocal microscopy, Optik 87:39–40.Google Scholar
  21. Wilson, T., and Carlini, A.R., 1989, The effect of aberrations on the axial response of confocal imaging systems, J. Microsc. 154:243–256.CrossRefGoogle Scholar
  22. Wilson, T., and Sheppard, C.J.R., 1984, Theory and Practice of Scanning Optical Microscopy, Academic Press, London.Google Scholar

Copyright information

© Springer Science+Business Media New York 1995

Authors and Affiliations

  • Stefan W. Hell
    • 1
  • Ernst H. K. Stelzer
    • 1
  1. 1.Light Microscopy Group, Cell Biophysics ProgrammeEuropean Molecular Biology Laboratory (EMBL)HeidelbergGermany

Personalised recommendations