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Proceedings Paper

Measured and computed point spread functions for an indirect water immersion objective used in three-dimensional fluorescence microscopy
Author(s): Nickos S. Kontoyannis; Frederick Lanni
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Paper Abstract

Living cells and other non-embedded biological specimens present a challenge in high- resolution microscopy because of their relatively low refractive index. It is well known that when oil-immersion objectives are used to look into these specimens, spherical aberration resulting from the index mismatch causes axial as well as transverse broadening of the 3D point spread function. Both effects reduce 3D resolution significantly, and cause the microscope to be axially shift-variant, thus complicating 3D deconvolution-based computational refinement. Axial shift variance should be much less a problem when water immersion objectives are used. With the aim of improving image data and our ability to refine it computationally, we have evaluated an apochromatic 40X/1.2 NA indirect water- immersion objective having an adjustable corrector for coverslip thickness. This work involves comparison of measured and computed 3D point spread functions, and comparison to the data acquired with an oil-immersion lens system. We show that focus-dependent spherical aberration is greatly reduced in the water-immersion system.

Paper Details

Date Published: 10 April 1996
PDF: 9 pages
Proc. SPIE 2655, Three-Dimensional Microscopy: Image Acquisition and Processing III, (10 April 1996); doi: 10.1117/12.237492
Show Author Affiliations
Nickos S. Kontoyannis, Carnegie Mellon Univ. (United States)
Frederick Lanni, Carnegie Mellon Univ. (United States)


Published in SPIE Proceedings Vol. 2655:
Three-Dimensional Microscopy: Image Acquisition and Processing III
Carol J. Cogswell; Gordon S. Kino; Tony Wilson, Editor(s)

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