10 April 1997 Comparison of 3D microscopy methods by imaging a well-characterized test object
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Proceedings Volume 2984, Three-Dimensional Microscopy: Image Acquisition and Processing IV; (1997) https://doi.org/10.1117/12.271274
Event: BiOS '97, Part of Photonics West, 1997, San Jose, CA, United States
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.
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James G. McNally, Carol J. Cogswell, Pal W. Fekete, Jose-Angel Conchello, "Comparison of 3D microscopy methods by imaging a well-characterized test object", Proc. SPIE 2984, Three-Dimensional Microscopy: Image Acquisition and Processing IV, (10 April 1997); doi: 10.1117/12.271274; https://doi.org/10.1117/12.271274
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