Re: wide field bead fluorescence

Posted by Daniel Possin on
URL: http://confocal-microscopy-list.275.s1.nabble.com/wide-field-bead-fluorescence-tp7582644p7582648.html

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An after thought: in the confocal microscope, given a pinhole size of 0.8 - 1.0 AU, the shell thickness should appear thinest when centrally focused on.  This is in fact a good test of one’s optical system.

Dan

On Sep 19, 2014, at 2:43 PM, Knecht, David <[hidden email]> wrote:

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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