wide field bead fluorescence

Posted by Knecht, David on
URL: http://confocal-microscopy-list.275.s1.nabble.com/wide-field-bead-fluorescence-tp7582644.html

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We have been imaging fluorescent beads in wide field and confocal microscopes.  The beads are 3 µm in diameter and surface labeled.  In the confocal, they look like doughnuts.  My expectation was that in wide field (60-100x 1.3-1.4 NA), they would look more uniformly labeled.  In reality, they look like doughnuts.  Can someone explain why?  

Dr. David Knecht
Professor of Molecular and Cell Biology
Core Microscopy Facility Director
University of Connecticut
Storrs, CT 06269
860-486-2200