Comparison of Wide-Field/Deconvolution and Confocal Microscopy for 3D Imaging

  • Peter J. Shaw


Optical microscopy has always been a central technique to biological research, but in recent years its importance has vastly increased, mainly because of the introduction of epifluorescence imaging, which gives very sensitive detection, coupled with a multitude of highly specific fluorescent probes. Furthermore, it is often possible to obtain useful images noninvasively at light levels that are not damaging to living cells. Microinjection and other cell-loading methods can therefore be used in combination with fluorescence microscopy to analyze and modify subcellular structure and function in living cells.


Spatial Frequency Focal Plane Point Spread Function Confocal Imaging Optical Section 
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  1. Agard, D. A., and Sedat, J.W., 1983, Three-dimensional architecture of apolytene nucleus, Nature 302:676–681.PubMedCrossRefGoogle Scholar
  2. Agard, D.A., Hiraoka, Y., Shaw, P.J., and Sedat J.W., 1989, Fluorescence microscopy in three dimensions, Methods Cell Biol. 30:353–378.PubMedCrossRefGoogle Scholar
  3. Aikens, R.S., Agard, D.A., and Sedat, J.W., 1989, Solid state imagers for microscopy, Methods Cell Biol. 29:291–313.PubMedCrossRefGoogle Scholar
  4. Carrington, W.A., Fogarty, K.E., and Fay, F.S., 1990, 3D fluorescence imaging of single cells using image restoration. In: Non-invasive Techniques in Cell Biology (Foskett and Grinstein, eds.), A.R. Liss, New York.Google Scholar
  5. Carter, K.C., Bowman, D., Carrington, W., Fogarty, K., McNeil, J.A., Fay, F.S., and Lawrence, J.B., 1993, A three-dimensional view of precursor messenger RNA metabolism within the mammalian nucleus, Science 259:1330–1335.PubMedCrossRefGoogle Scholar
  6. Castleman, K.R., 1979, Digital Image Processing, Prentice-Hall, Englewood Cliffs, New Jersey.Google Scholar
  7. Erhardt, A., Zinser, G., Komitowski, D., and Bille, J., 1985, Reconstructing 3D light microscopic images by digital image processing, Appl. Opt. 24:194.PubMedCrossRefGoogle Scholar
  8. Fay, F.S., Carrington, W., andFogarty, K.E., 1989, Three-dimensional molecular distribution in single cells analyzed using a digital imaging microscope, J. Microsc, 153 (pt 2): 133–149.PubMedCrossRefGoogle Scholar
  9. Flanders, D.J., Rawlins, D.J., Shaw, P.J., and Lloyd, C.W., 1990, Re-establishment of the interphase microtubule array in vacuolated plant cells, studied by confocal microscopy and 3-D imaging, Development 110:897–904.Google Scholar
  10. Frieden, B.R., 1984, Maximum-likelihood estimates of spectra. In: Deconvolu-tion with Applications in Spectroscopy (P.A. Jansson, ed), Academic Press, New York, pp. 229–261.Google Scholar
  11. Highett, M.I., Rawlins, D.J., and Shaw, P.J., 1993a, Different patterns of rDNA distribution in Pisum sativum nucleoli correlate with different levels of nucleolar activity, J. Cell Sci. 104:843–852.Google Scholar
  12. Highett, M.I., Beven, A.F., and Shaw, P.J., 1993b, Localization of 5S genes and transcripts in Pisum sativum nuclei, J. Cell Sci. 105:1151–1158.PubMedGoogle Scholar
  13. Hiraoka, Y., Sedat, J.W., and Agard, D.A., 1988, The use of a charge-coupled device for quantitative optical microscopy of biological structures, Science 238:36–41.CrossRefGoogle Scholar
  14. Hiraoka, Y., Minden, J.S., Swedlow, J.R., Sedat, J.W., and Agard, D.A., 1989, Focal points for chromosome condensation and decondensation from three-dimensional in vivo time-lapse microscopy, Nature 342:293–296.PubMedCrossRefGoogle Scholar
  15. Hiraoka, Y., Sedat, J.W., and Agard, D.A., 1990, Determination of three-dimensional imaging properties of a light microscope system: Partial confocal behaviour in epifluorescence microscopy, Biophys. J. 57:325–333.PubMedCrossRefGoogle Scholar
  16. Holmes, T.J., 1988, Maximum-likelihood restoration adapted for noncoherent optical imaging, J. Opt. Soc. Am. A5:666–673.CrossRefGoogle Scholar
  17. Holmes, T.J., and Liu, Y.-H., 1992, Image restoration for 2-D and 3-D fluorescence microscopy. In: Visualization in Biomedical Microscopies (A. Kriete, ed), VCH, Weinheim, Germany.Google Scholar
  18. Inoué, S., 1986, Video Microscopy, Plenum Press, New York.Google Scholar
  19. Jansson, P.A., Hunt, R.M., and Plyler, E.K., 1970, J. Opt. Soc. Am. 60:596.CrossRefGoogle Scholar
  20. Pawley, J.B., 1994, The sources of noise in three-dimensional microscopical data sets. In: Three-dimensional Confocal Microscopy: Volume Investigation of Biological Specimens (J.K. Stevens, L.R. Mills, and J.E. Trogadis, eds.), Academic Press, New York, pp. 48–94.Google Scholar
  21. Pawley, J.B., and Smallcomb, A., 1992, An introduction to practical confocal microscopy: The ultimate form of biological light microscopy? Acta Microsc. 1:58–73.Google Scholar
  22. Sandison, D.R., Piston, D.W., and Webb, W.W., 1994, Background rejection and optimization of signal-to-noise in confocal microscopy. In: Three-Dimensional Confocal Microscopy: Volume Investigation of Biological Specimens (J.K Stevens, L.R. Mills, and J. E Trogadis., eds), Academic Press, New York, pp. 29–47.CrossRefGoogle Scholar
  23. Self, S.A., 1983, Focusing of spherical Gaussian beams, Appl. Opt. 22:658–661.PubMedCrossRefGoogle Scholar
  24. Shaw, P.J., 1993, Computer reconstruction in three-dimensional fluorescence microscopy. In: Electronic Light Microscopy (D. Shotton, ed), Wiley-Liss, New York, pp. 211–230.Google Scholar
  25. Shaw, P.J., and Rawlins, D.J., 1991a, Three-dimensional fluorescence microscopy, Prog. Biophys. Molec. Biol. 56:187–213.CrossRefGoogle Scholar
  26. Shaw, P.J., and Rawlins, D.J., 1991b, The point spread function of a confocal microscope: Its measurement and use in deconvolution of 3D data, J. Microsc. 163:151–165.CrossRefGoogle Scholar
  27. Sheppard, C.J.R., and Choudhury, A., 1977, Image formation in the scanning microscope, Opt. Acta. 24:1051–1073.CrossRefGoogle Scholar
  28. Stokseth, P.A., 1969, Properties of a defocused optical system, J. Opt. Soc. Am. 59:1314–1321.CrossRefGoogle Scholar
  29. Wilson, T., 1993, Image formation in confocal microscopy. In: Electronic Light Microscopy (D.M. Shotton, ed), Wiley-Liss, New York.Google Scholar
  30. Young, I.T., 1989, Image fidelity: Characterizing the imaging transfer function, Methods Cell Biol. 30:2–47.Google Scholar
  31. Zhang, D.H., Wadsworth, P., and Hepler, P.K., 1990, Microtubule dynamics in living dividing plant cells: Confocal imaging of microinjected fluorescent brain tubulin, Proc. Natl Acad. Sci. USA. 87:8820–8824.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1995

Authors and Affiliations

  • Peter J. Shaw
    • 1
  1. 1.Department of Cell BiologyJohn Innes InstituteColney Lane, NorwichUK

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