Fundamental Limits in Confocal Microscopy

  • James Pawley

Abstract

The previous chapter described how the confocal approach developed from conventional light microscopy and outlined the basic advantages to be gained by the use of confocal sampling techniques. This chapter will discuss the fundamental considerations that limit the performance of all confocal microscopes. Though at present no commercially available equipment approaches these limits, some simple tests will be described to help the user assess how closely a given instrument performs. Additional information to help the user to operate the confocal microscope in an optimal manner can be found in Chapter 36, “Tutorial on Practical Confocal Microscopy and Use of the Confocal Test Specimen,” which also includes methods for measuring resolution.

Keywords

Confocal Laser Scanning Microscope Numerical Aperture Photon Efficiency Contrast Transfer Function Field Diaphragm 
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.

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References

  1. Gelles, J., Schnapp, B.J., Steur, E., and Scheetz, M.P., 1988, Nanometer scale motion analysis of microtubule-based motor enzymes, Proc. EMSA 46:68–69.Google Scholar
  2. Gunter, W.D. Jr., Grant, G.R., and Shaw, S., 1970, Optical devices to increase photocathode quantum efficiency, Appl. Optics 9(2):251–257.CrossRefGoogle Scholar
  3. Oldenbourg, R., Terada, H., Tiberio, R., and Inoué, S., 1993, Image sharpness and contrast transfer in coherent confocal microscopy, J. Microsc. 172:31–39.PubMedCrossRefGoogle Scholar
  4. Paddy, M.R., Hiraoka, Y., Chen, H., Sedat, J.W., and Agard, D.A., 1988, A structural overview of the nucleus as derived from CCD-based 3-D optical microscopy, Proc. EMSA 46:34–35.Google Scholar
  5. Pawley, J.B., 1994, The sources ofnoise in three-dimensional microscopical data sets. In: Three Dimensional Confocal Microscopy: Volume Investigation of Biological Specimens (J. Stevens, ed.), Academic Press, New York, pp. 47–94.CrossRefGoogle Scholar
  6. Pawley, J.B., and Centonze, V., 1994, Practical laser-scanning confocal light microscopy: Obtaining optimal performance from your instrument. In: Cell Biology: A Laboratory Handbook, (J.E. Celis, ed.), Academic Press, New York, Vol. 2, pp. 44–64.Google Scholar
  7. Pawley, J.B., Amos, W.B., Dixon, A., and Brelje, T.C., 1993a, Simultaneous, non-interfering, collection of optimal fluorescent and backscattered light signals on the MRC-500/600, Proc. Microsc. Soc. Am. 51:156–157.Google Scholar
  8. Pawley, J.B., Hasko, D., and Cleaver, J., 1993b, A standard test and calibration specimen for confocal microscopy II. In: Proceedings of the 1993 International Conference on Confocal Microscopy and 3-D Image Processing, Sydney, Australia (C.J.R. Sheppard, ed.), p. 35d.Google Scholar
  9. Pawley, J.B., Wright, A.G., and Garrard, C.C., 1993c, Optical enhancement and pulse-counting improve the quality of confocal data. In: Proceedings of the 1993 International Conference on Confocal Microscopy and 3-D Image Processing, Sydney, Australia (C.J.R. Sheppard, ed.), p. 69.Google Scholar
  10. Pollock, H.N., and Chapman, D.S., 1993, Underground records of changing climate, Sci. Am. 68:44–50.CrossRefGoogle Scholar
  11. Rose, A., 1948, Television pickup tubes and the problem of noise, Adv. Electron. 1:131.Google Scholar
  12. Sandison, D.R., Piston, D.W., and Webb, W.W., 1994, Background rejection and optimization of signal to noise ratio in confocal microscopy. In: Three-Dimensional Confocal Microscopy: Volume Investigations of Biological Systems (J.K. Stevens, L.R. Mills, and J.E. Trogadis, eds.), Academic Press, New York, pp. 29–48.CrossRefGoogle Scholar
  13. Sheppard, J. R., Gu, M., and Roy, M., 1992, Signal-to-noise ratio in confocal microscope systems, J. Microsc. 168:209–218.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1995

Authors and Affiliations

  • James Pawley
    • 1
  1. 1.Zoology DepartmentUniversity of WisconsinMadisonUSA

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