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Applied Optics

Applied Optics

APPLICATIONS-CENTERED RESEARCH IN OPTICS

  • Vol. 33, Iss. 4 — Feb. 1, 1994
  • pp: 603–615

Optics InfoBase > Applied Optics > Volume 33 > Issue 4 > Background rejection and signal-to-noise optimization in confocal and alternative fluorescence microscopes

Background rejection and signal-to-noise optimization in confocal and alternative fluorescence microscopes

David R. Sandison and Watt W. Webb  »View Author Affiliations


Applied Optics, Vol. 33, Issue 4, pp. 603-615 (1994)
http://dx.doi.org/10.1364/AO.33.000603


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Abstract

In the confocal microscope, tightly focused illumination and spatially filtered detection are combined to reduce out-of-focus background and to produce high-quality images that display thin optical sections within thick fluorescent specimens. We define background as the detected light that originates outside a resolution volume and signal as the detected light that originates within the same volume. Background rejection is measured by the signal-to-background ratio (S/B) and is calculated for confocal, spinning-disk, line-illumination, slit-detection, and conventional fluorescence microscopes as a function of both the spatial filter size and the specimen thickness. Spatial filter sizes that reject background and optimize the signal-to-noise ratio (S/N) are calculated for each microscope. These calculations are normalized so that the time-averaged illumination at each point in the specimen is the same for each microscope. For thick specimens, we show that the S/B obtained with a confocal microscope can be more than 100 times greater than the S/B available with a conventional microscope, and we find that the optimal confocal S/N can be a factor of 10 greater than the S/N in the conventional microscope.

© 1994 Optical Society of America

History
Original Manuscript: March 25, 1993
Revised Manuscript: September 8, 1993
Published: February 1, 1994

Citation
David R. Sandison and Watt W. Webb, "Background rejection and signal-to-noise optimization in confocal and alternative fluorescence microscopes," Appl. Opt. 33, 603-615 (1994)
http://www.opticsinfobase.org/ao/abstract.cfm?URI=ao-33-4-603


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References

  1. M. Minsky, “Microscopy apparatus,” U.S. patent3,013,467 (19December1961).
  2. M. Bendinelli, A. Consortini, L. Ronchi, B. R. Frieden, “Degrees of freedom and eigenfunctions for the noisy image,” J. Opt. Soc. Am. 64, 1498–1502 (1974). [CrossRef]
  3. I. J. Cox, C. J. R. Sheppard, “Information capacity and resolution in an optical system,” J. Opt. Soc. Am. A 3, 1152–1158 (1986). [CrossRef]
  4. T. Wilson, C. J. R. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, New York, 1984).
  5. T. Wilson, Confocal Microscopy (Academic, San Diego, Calif., 1990).
  6. S. Kimura, C. Munakata, “Calculation of three-dimensional optical transfer function for a confocal scanning fluorescent microscope,” J. Opt. Soc. Am. A 6, 1015–1019 (1989). [CrossRef]
  7. W. W. Webb, K. Wells, D. R. Sandison, J. Strickler, Optical Microscopy for Biology (Wiley-Liss, New York, 1990), pp. 73–108.
  8. C. J. R. Sheppard, C. J. Cogswell, M. Gu, “Signal strength and noise in confocal microscopy: factors influencing selection of an optimum detector aperture,” Scanning 13, 233–240 (1991). [CrossRef]
  9. C. J. R. Sheppard, “Stray light and noise in confocal microscopy,” Micron Microsc. Acta 22, 239–243 (1991). [CrossRef]
  10. K. S. Wells, D. R. Sandison, J. Strickler, W. W. Webb, “Quantitative fluorescence imaging with laser scanning confocal microscopy,” in Handbook of Biological Confocal Microscopy, J. B. Pawley, ed. (Plenum, New York, 1990), Chap. 3. [CrossRef]
  11. D. R. Sandison, D. W. Piston, W. W. Webb, “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, J. E. Trogadis, eds., (Academic, New York, 1994), Chap. 2.
  12. M. D. Egger, M. Petràň, “New reflected light microscope for viewing unstained brain and ganglion cells,” Science 157, 305–307(1967). [CrossRef] [PubMed]
  13. G. S. Kino, “Intermediate optics in Nipkow disk microscopes,” in Handbook of Biological Confocal Microscopy, J. B. Pawley, ed. (Plenum, New York, 1990), Chap. 10. [CrossRef]
  14. C. J. Koester, “A scanning mirror microscope with optical sectioning characteristics: applications in ophthalmology,” Appl. Opt. 19, 1749–1757 (1980). [CrossRef] [PubMed]
  15. G. J. Brakenhoff, K. Visscher, “Novel confocal imaging and visualization techniques,” in Micro 90: Proceedings of the Royal Microscopical Society Conference, H. Y. Elder, ed. (Hilger, London, 1990), Chap. 9.
  16. J. W. Lichtman, W. J. Sunderland, R. S. Wilkinson, “High-resolution imaging of synaptic structure with a simple confocal microscope,” New Biologist 1, 75–82 (1989). [PubMed]
  17. T. Wilson, C. J. R. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, New York, 1984), p. 3.
  18. M. Born, E. Wolf, Principles of Optics, 6th ed. (Pergamon, New York, 1980), p. 437.
  19. T. Wilson, C. J. R. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, New York, 1984), p. 25.
  20. C. J. R. Sheppard, H. J. Matthews, “Imaging in high-aperture optical systems,” J. Opt. Soc. Am. A 4, 1354–1360 (1987). [CrossRef]
  21. Documentation and FORTRAN code for Hankel Transform Library kindly supplied and supported by R. F. Loane, Department of Applied and Engineering Physics, Cornell University, Ithaca, N. Y.
  22. F. Johnson, “An improved method for computing a discrete Hankel transform,” Comput. Phys. Commun. 43, 181–202 (1987). [CrossRef]
  23. T. Wilson, C. J. R. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, New York, 1984), p. 33.
  24. D. R. Sandison, “Fluorescence confocal laser scanning microscopy for three-dimensional imaging of living biological specimens,” Ph.D. dissertation (Cornell University, Ithaca, N. Y., 1993), p. 97.
  25. Y. Hiraoka, J. W. Sedat, D. A. Agard, “Determination of three-dimensional imaging properties of a light microscope system,” Biophys. J. 57, 325–333 (1990). [CrossRef] [PubMed]
  26. W. A. Carrington, K. E. Fogarty, L. Lifschitz, F. S. Fay, “Three-dimensional imaging on confocal and wide-field microscopes,” in Handbook of Biological Confocal Microscopy, J. B. Pawley, ed. (Plenum, New York, 1990), Chap. 14. [CrossRef]
  27. I. S. Gradshteyn, I. M. Ryzhik, Table of Integrals, Series, and Products (Academic, New York, 1980), integral 6.554.3, p. 682.

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