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I think it's because a larger surface area of a bead is contained in a cross section closer to the edges than in the middle.

Mike
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From: Confocal Microscopy List <[log in to unmask]> on behalf of Jeremy Adler <[log in to unmask]>
Sent: Saturday, September 20, 2014 12:13 AM
To: [log in to unmask]
Subject: Re: wide field bead fluorescence

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1) Surface labeling is the key - consider all possible horizontal optical slices, then sum the slices and blur them a little - as a wide field microscope does.

2) Refractive index mismatch is also likely - what are they mounted in ?. From memory microspheres have a RI near that of oil.

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From: Confocal Microscopy List [[log in to unmask]] on behalf of Knecht, David [[log in to unmask]]
Sent: 19 September 2014 23:43
To: [log in to unmask]
Subject: wide field bead fluorescence

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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