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Applied Optics

Applied Optics


  • Vol. 41, Iss. 4 — Feb. 1, 2002
  • pp: 685–690

Influence of refractive-index mismatch in high-resolution three-dimensional confocal microscopy

Alberto Diaspro, Federico Federici, and Mauro Robello  »View Author Affiliations

Applied Optics, Vol. 41, Issue 4, pp. 685-690 (2002)

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The effects of the refractive-index mismatch in confocal laser scanning microscopy were extensively studied. The axial aberration induced in the case of fluorescent microspheres was measured. The data were used to take into account the mismatch-induced aberrations and to consider object-size influence. Then we focused on the effect of refractive-index mismatch on the effective system’s point-spread function under different mismatch conditions and on depth of focusing. We experimentally verified that the peak of the point-spread function intensity profile decreases and the point-spread function itself progressively broadens as a function of the combined effect of the refractive-index mismatch and of the penetration depth, leading to a worsening of the system’s overall performances. We also performed these same measurements by embedding subresolution beads in an oocyte’s cytoplasm, which can be considered a turbid medium. We found evidence consistent with the previously developed theoretical model; in particular we found a strong dependence of the intensity peak on the focusing depth.

© 2002 Optical Society of America

OCIS Codes
(080.1010) Geometric optics : Aberrations (global)
(080.2710) Geometric optics : Inhomogeneous optical media
(170.3880) Medical optics and biotechnology : Medical and biological imaging
(180.1790) Microscopy : Confocal microscopy
(180.2520) Microscopy : Fluorescence microscopy
(180.6900) Microscopy : Three-dimensional microscopy

Original Manuscript: April 13, 2001
Revised Manuscript: August 6, 2001
Published: February 1, 2002

Alberto Diaspro, Federico Federici, and Mauro Robello, "Influence of refractive-index mismatch in high-resolution three-dimensional confocal microscopy," Appl. Opt. 41, 685-690 (2002)

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