Comparison of 3D microscopy methods by imaging a well-characterized test object

James G. McNally; Carol J. Cogswell; Pal W. Fekete; Jose-Angel Conchello

doi:10.1117/12.271274

10 April 1997 Comparison of 3D microscopy methods by imaging a well-characterized test object

James G. McNally, Carol J. Cogswell, Pal W. Fekete, Jose-Angel Conchello

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.

Citation Download Citation

James G. McNally, Carol J. Cogswell, Pal W. Fekete, and 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); https://doi.org/10.1117/12.271274

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