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Journal of the Optical Society of America A

Journal of the Optical Society of America A


  • Vol. 9, Iss. 1 — Jan. 1, 1992
  • pp: 154–166

Experimental test of an analytical model of aberration in an oil-immersion objective lens used in three-dimensional light microscopy

Sarah Frisken Gibson and Frederick Lanni  »View Author Affiliations

JOSA A, Vol. 9, Issue 1, pp. 154-166 (1992)

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Oil-immersion microscope objective lenses have been designed and optimized for the study of thin, two-dimensional object sections that are mounted immediately below the coverslip in a medium that is index matched to the immersion oil. It has been demonstrated both experimentally and through geometrical- and physical-optics theory that, when the microscope is not used with the correct coverslip or immersion oil, when the detector is not located at the optimal plane in image space, or when the object does not satisfy specific conditions, aberration will degrade both the contrast and the resolution of the image. In biology the most severe aberration is introduced when an oil-immersion objective lens is used to study thick specimens, such as living cells and tissues, whose refractive indices are significantly different from that of the immersion oil. We present a model of the three-dimensional imaging properties of a fluorescence light microscope subject to such aberration and compare the imaging properties predicted by the model with those measured experimentally. The model can be used to understand and compensate for aberration introduced to a microscope system under nondesign optical conditions so that both confocal laser scanning microscopy and optical serial sectioning microscopy can be optimized.

© 1992 Optical Society of America

Original Manuscript: July 11, 1990
Revised Manuscript: April 8, 1991
Manuscript Accepted: April 9, 1991
Published: January 1, 1992

Sarah Frisken Gibson and Frederick Lanni, "Experimental test of an analytical model of aberration in an oil-immersion objective lens used in three-dimensional light microscopy," J. Opt. Soc. Am. A 9, 154-166 (1992)

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  15. The accuracy of this assumption can easily be verified for a microscope lens system by the use of a planar diffraction grating object illuminated by a monochromatic, collimated laser and a telescope used to observe the back focal surface. We made these measurements on the microscope objective lenses described in this paper and found that the lenses produced compact images over the back focal surface of the individual diffraction orders formed by the grating and that this surface was planar to ±100 μm across the diameter of the back focal surface. The compact image of each order had the appearance of an Airy pattern, from which we conclude that each is a nearly stigmatic image of the incoming parallel rays. We also verified that the diffraction orders were equispaced in the focal plane, as is expected for a lens that obeys the sine condition. The results of this experiment verify the assumption that parallel rays entering a lens system will travel equal optical paths and cross in the back focal plane as well as the assumption that the back focal surface is planar.
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