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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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