Analysis of spherical aberration of a water immersion objective: application to specimens with refractive indices 1.33–1.40

Authors

  • D.-S. Wan,

    1. Wellman Laboratory of Photomedicine, Department of Dermatology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, U.S.A.,
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  • M. Rajadhyaksha,

    1. Wellman Laboratory of Photomedicine, Department of Dermatology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, U.S.A.,
    2. Lucid Technologies, Henrietta, New York, U.S.A.
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  • R. H. Webb

    1. Wellman Laboratory of Photomedicine, Department of Dermatology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, U.S.A.,
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Abstract

The method of using immersion medium to correct spherical aberration for water immersion objectives when the samples are not water is investigated. Spherical aberration is measured by an interferometer converted from a confocal microscope for samples with different refractive indices. When the proper refractive index of the immersion medium and thickness of cover slip are selected, the measured spherical aberration approaches zero. A theoretical model can be used for prediction of the immersion medium to correct spherical aberration for various samples. Using the thinnest available cover slip (100 μm), the zero spherical aberration condition can be applied to samples with refractive index as high as 1.40. Confocal images in the condition of almost no spherical aberration are included to demonstrate the improvement of axial resolution due to this correction.

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