wide field bead fluorescence

[Knecht, David]

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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

[Reto Fiolka]

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Hi David

Maybe because the extend of the PSF (widefield or confocal) is about 4 times larger axially
than laterally.

Thus the convolution of a shell (bead) with a cigar (PSF) might yield an intensity distribution
that looks a bit like a toroid.

Further, if your focal plane coincides with the center of the sphere, most signal will be
detected from the segments of the shell that intersect the focal plane or are close by. Since
the optical axis is tangential to the local shell surface there, more signal is integrated by the
axially extended PSF. The top and bottom part of the shell are already slightly out of the
depth of focus and will be detected more weakly. Even if they fall into the depth of focus,
the optical axis is normal to the surface there, and only a thin layer falls into the PSF.

Hence the drop off in the center.


Best,
Reto

[Daniel Possin]

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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

[Jeremy Adler-4]

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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.

________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Knecht, David [[hidden email]]
Sent: 19 September 2014 23:43
To: [hidden email]
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

[mmodel]

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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
________________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Jeremy Adler <[hidden email]>
Sent: Saturday, September 20, 2014 12:13 AM
To: [hidden email]
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.

________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Knecht, David [[hidden email]]
Sent: 19 September 2014 23:43
To: [hidden email]
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

[James Pawley]

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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:
>
>>  *****
>>  To join, leave or search the confocal microscopy listserv, go to:
>>  http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>  Post images on http://www.imgur.com and include the link in your posting.
>>  *****
>>
>>  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

--
               ****************************************
James and Christine Pawley, 5446 Burley Place (PO
Box 2348), Sechelt, BC, Canada, V0N3A0,
Phone [phone], email <[hidden email]>
NEW! NEW! AND DIFFERENT Cell (when I remember to turn it on!) 1-604-989-6146

[Knecht, David]

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On Sep 20, 2014, at 8:12 AM, James Pawley <[hidden email]> wrote:

> *****
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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?)?
fluorescent protein bound to the surface of a latex bead so molecularly thin on the scale of an optical  microscope.
> Are we correct in assuming that the WF focus plane coincides with the centre of the bead?
yes
> What is the RI of the beads and the embedding medium?
live imaging of beads inside phagosomes inside cells with oil immersion and cells in media using a glass bottom dish
> 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.
related to this issue, it is not clear to me the extent to which latex beads are transparent and that would be one explanation if excitation or emission is restricted by the inability of light from the focal planes above the middle getting through the lower part of the bead to get back to the objective.

>
> 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
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your posting.
>> *****
>>
>> 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:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> Post images on http://www.imgur.com and include the link in your posting.
>>> *****
>>>
>>> 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
>
>
> --
>             ****************************************
> James and Christine Pawley, 5446 Burley Place (PO Box 2348), Sechelt, BC, Canada, V0N3A0,
> Phone [phone], email <[hidden email]>
> NEW! NEW! AND DIFFERENT Cell (when I remember to turn it on!) 1-604-989-6146

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

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

[Vitaly Boyko]

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Dear All,

Would someone know if the Leica LAS-Reader function is available in public domain written either for matlab or dipimage?

Thank you,

Vitaly