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Proceedings Paper

Comparison of 3D microscopy methods by imaging a well-characterized test object
Author(s): James G. McNally; Carol J. Cogswell; Pal W. Fekete; Jose-Angel Conchello
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Paper Abstract

We used a 10-micrometer-diameter fluorescent bead as a test object for 3D microscopy, and independently determined its structure by examining 1-micrometer-thick physical sections of the bead. Images of the full bead on different 3D microscopes revealed a number of aberrations and distortions. Images also showed evidence for absorption and/or scattering on all confocal and wide-field microscopes tested, but not on a two-photon microscope. The best 3D images of the bead came from deconvolution of either wide- field or certain confocal images. For deconvolution of partially confocal data, a region extending 7 micrometers beyond the top and bottom of the bead contained out-of- focus-light information essential for correct restoration. Fully confocal images required 10-fold less computer time for deconvolution, but 1000-fold more excitation light than a wide-field image, even though the resultant restorations were reasonably comparable. Laser imaging of the bead appeared to produce an artifactual image for which we currently have no explanation.

Paper Details

Date Published: 10 April 1997
PDF: 12 pages
Proc. SPIE 2984, Three-Dimensional Microscopy: Image Acquisition and Processing IV, (10 April 1997); doi: 10.1117/12.271274
Show Author Affiliations
James G. McNally, Washington Univ. (United States)
Carol J. Cogswell, Univ. of Sydney (United States)
Pal W. Fekete, Univ. of Sydney (Australia)
Jose-Angel Conchello, Washington Univ. (United States)

Published in SPIE Proceedings Vol. 2984:
Three-Dimensional Microscopy: Image Acquisition and Processing IV
Carol J. Cogswell; Jose-Angel Conchello; Tony Wilson, Editor(s)

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