Re: wide field bead fluorescence

Posted by James Pawley on
URL: http://confocal-microscopy-list.275.s1.nabble.com/wide-field-bead-fluorescence-tp7582644p7582652.html

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Hi David,

Could you tell us a little more? How thick is the
"surface label" (molecular or a layer of
plastic?)? Are we correct in assuming that the WF
focus plane coincides with the centre of the
bead? What is the RI of the beads and the
embedding medium? Using NA 1.3-1.4 implies "all
oil or glass to the focus plane". Anything else
involves all sorts of refraction, reflection
games near the bead surface.

As a not-too-close analogy, it has recently been
recognized that, if you really fill the BFP of
such an objective with excitation light when
viewing a watery specimen, you get enhanced
excitation right near the glass/water interface
from the TIRF cause by the high-NA being (mostly)
reflected at this interface.

More prosaically, light created in a high RI
plastic bead my have trouble getting out because
of total or partial reflections. For instance,
more of it than we would expect may leak out near
where the bead touches the glass as this is a
break in the RI barrier.

If the bead is in oil and has about the same RI
as oil, then I guess that I would expect that
when the focus plane passes through the centre of
the bead, you would record a doughnut in both
confocal and WF. Let's assume that the
fluorescent layer is very thin. In WF, the whole
surface will be excited about evenly (not true
for a plastic bead in water) but the light
emitted from the upper and lower surface will be
1.5 µm out of focus at the image plane and at
NA1.3 that is a long way. The light will make it
to the CCD but it will be blurred over a larger
area and so look dimmer. Although not as much
dimmer as the confocal image where you have two
PSFs to make it dimmer.

Best,

Jim Pawley

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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

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