field flatness Yokogawa W1-Borealis

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Dear List,

We are having a problem with field flatness on our Yokogawa-W1-Borealis Unit using Andor 888’s as cameras. We measure a drop of intensity of about 60% from the middle to the sides after a rigid adapter was installed between the stand and the unit.
Did anyone encounter similar problems or does anyone maybe have a service manual?

Thank you!

Jens

[James Jonkman-2]

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Jens, maybe you know this already but you can't use a thick chroma slide to measure uniformity on a spinning disk due to pinhole crosstalk.  When I first got my Borealis update it looked terrible with a Chroma slide, but when I switched to a saturated dye solution it showed excellent uniformity.  Or you can test it by imaging a thin slide (Molecular Probes prepared slide #1 for example) and moving a feature from the middle to the corner.  The saturated dye solution is a neat idea (first heard about it from John Oreopoulos probably on this listserv - he is now with Andor).  Just put concentrated FITC in a coverglass bottom dish, and it quenches everywhere except in a thin layer at the surface thereby creating a thin fluorescent layer that doesn't photobleach (diffusion takes away the photobleached molecules).

If the non-uniformity is truly messed up then I look forward to hearing how you solve it.  good luck!

Cheers,
James


-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Jens Bernhard Bosse
Sent: June 26, 2019 11:06 AM
To: [hidden email]
Subject: field flatness Yokogawa W1-Borealis

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Dear List,

We are having a problem with field flatness on our Yokogawa-W1-Borealis Unit using Andor 888’s as cameras. We measure a drop of intensity of about 60% from the middle to the sides after a rigid adapter was installed between the stand and the unit.
Did anyone encounter similar problems or does anyone maybe have a service manual?

Thank you!

Jens

[Mark Cannell-2]

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Vignetting can occur when camera sensor is moved further away from the design position (it's due to clipping of marginal rays). You may be able to look at the image on a sheet of paper at the camera sensor position with bright field illumination to try to find what is clipping? To fix it you may need to change coupling lens strength and field size.

HTH

Mark B. Cannell. Ph.D. FRSNZ FISHR
Department of Physiology, Pharmacology & Neuroscience
School of Medical Sciences
University Walk
Bristol BS8 1TD
 
[hidden email]
 
 

On 26/06/19, 4:22 PM, "Confocal Microscopy List on behalf of Jens Bernhard Bosse" <[hidden email] on behalf of [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.
    *****
   
    Dear List,
   
    We are having a problem with field flatness on our Yokogawa-W1-Borealis Unit using Andor 888’s as cameras. We measure a drop of intensity of about 60% from the middle to the sides after a rigid adapter was installed between the stand and the unit.
    Did anyone encounter similar problems or does anyone maybe have a service manual?
   
    Thank you!
   
    Jens

[BROWNE Mark]

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Commercial response.

Jens
I am intrigued as to why a rigid adapter was installed? Can you expand - possibly off-line if you wish. We will try to assist you recover the performance.

Best regards
Mark Browne,

[hidden email]

Sent from my iPhone

> On Jun 26, 2019, at 4:40 PM, Mark Cannell <[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.
> *****
>
> Vignetting can occur when camera sensor is moved further away from the design position (it's due to clipping of marginal rays). You may be able to look at the image on a sheet of paper at the camera sensor position with bright field illumination to try to find what is clipping? To fix it you may need to change coupling lens strength and field size.
>
> HTH
>
> Mark B. Cannell. Ph.D. FRSNZ FISHR
> Department of Physiology, Pharmacology & Neuroscience
> School of Medical Sciences
> University Walk
> Bristol BS8 1TD
>
> [hidden email]
>
>
>
> On 26/06/19, 4:22 PM, "Confocal Microscopy List on behalf of Jens Bernhard Bosse" <[hidden email] on behalf of [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.
>    *****
>
>    Dear List,
>
>    We are having a problem with field flatness on our Yokogawa-W1-Borealis Unit using Andor 888’s as cameras. We measure a drop of intensity of about 60% from the middle to the sides after a rigid adapter was installed between the stand and the unit.
>    Did anyone encounter similar problems or does anyone maybe have a service manual?
>
>    Thank you!
>
>    Jens
>

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[patrick deguelle]

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If the rigid adapter that was placed between the microscope changed the distance between the scan head and the microscope, a different field lens on the Yokogawa would likely have to be utilized.  If the distance between the Yokogawa and microscope is the same, then it is most certainly an alignment issue.  My guess is that you have a combination of both issues.

Sincerely,

Patrick De Guelle

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________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Mark Cannell <[hidden email]>
Sent: Wednesday, June 26, 2019 4:40:03 PM
To: [hidden email]
Subject: Re: field flatness Yokogawa W1-Borealis

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Vignetting can occur when camera sensor is moved further away from the design position (it's due to clipping of marginal rays). You may be able to look at the image on a sheet of paper at the camera sensor position with bright field illumination to try to find what is clipping? To fix it you may need to change coupling lens strength and field size.

HTH

Mark B. Cannell. Ph.D. FRSNZ FISHR
Department of Physiology, Pharmacology & Neuroscience
School of Medical Sciences
University Walk
Bristol BS8 1TD

[hidden email]



On 26/06/19, 4:22 PM, "Confocal Microscopy List on behalf of Jens Bernhard Bosse" <[hidden email] on behalf of [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.
    *****

    Dear List,

    We are having a problem with field flatness on our Yokogawa-W1-Borealis Unit using Andor 888’s as cameras. We measure a drop of intensity of about 60% from the middle to the sides after a rigid adapter was installed between the stand and the unit.
    Did anyone encounter similar problems or does anyone maybe have a service manual?

    Thank you!

    Jens

[John Oreopoulos]

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Credit must be given to Mike Model (also very active on this listserv) for conceiving the idea of using a concentrated dye solution as a microscope diagnostic sample. He first published about them almost 2 decades ago:

https://www.ncbi.nlm.nih.gov/pubmed/11500847

and subsequently produced other publications showing how these samples can be used for other useful quantitative imaging purposes. A thick fluorescent plastic specimen is probably suitable for measuring field flatness or uniformity on a laser-scanning confocal microscope, but for a spinning disk confocal microscope, it produces the worst possible (and unrealistic) case of complete and total pinhole cross-talk which superimposes the illumination profile. As mentioned by James, the beauty of the concentrated dye solutions - besides their cheapness, ease of creation, and spectral variety - is that they only produce fluorescence from a diffraction-limited layer adjacent to the coverslip due to their optical density. An example of the difference can be seen here, where a maximum intensity z-projection of plastic slide and dye slide acquired using Andor's Borealis illumination technique on a spinning disk confocal system are presented:

https://drive.google.com/file/d/1vOP0TxLCDAJYVMlKe7dQSv7XxZLEEWfy/view?usp=sharing

In the example above, the illumination was purposefully reduced to be a sub-section of the the entire camera field of view to show the pinhole cross-talk effect.

Not only does such a specimen allow one to measure the true illumination profile on a spinning disk confocal microscope, but a z-scan will also reveal other useful metrics, such as the thickness of the optical section. They can also be used for flat-field/shading correction, and Kurt Thorn wrote a nice piece on this in his imaging blog:

http://nic.ucsf.edu/blog/2014/01/shading-correction-of-fluorescence-images/
http://nic.ucsf.edu/blog/2014/01/fluorescent-dyes-for-shading-correction/
http://nic.ucsf.edu/blog/2014/04/shading-correction-for-different-objectives-and-channels/

But it turns out that the story on achieving images free of non-uniformity artifacts is more complicated. Making sure the illumination light coming out of the confocal scan head is flat is just one aspect of the situation; How the scan head mates to the microscope and funnels the laser light to the objective lens, and the chromatic aberrations of the objective lens itself play an equally important role, especially on systems using large field of view sCMOS cameras.

John Oreopoulos

On 2019-06-26, at 1:13 PM, BROWNE Mark wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
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> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Commercial response.
>
> Jens
> I am intrigued as to why a rigid adapter was installed? Can you expand - possibly off-line if you wish. We will try to assist you recover the performance.
>
> Best regards
> Mark Browne,
>
> [hidden email]
>
> Sent from my iPhone
>
>> On Jun 26, 2019, at 4:40 PM, Mark Cannell <[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.
>> *****
>>
>> Vignetting can occur when camera sensor is moved further away from the design position (it's due to clipping of marginal rays). You may be able to look at the image on a sheet of paper at the camera sensor position with bright field illumination to try to find what is clipping? To fix it you may need to change coupling lens strength and field size.
>>
>> HTH
>>
>> Mark B. Cannell. Ph.D. FRSNZ FISHR
>> Department of Physiology, Pharmacology & Neuroscience
>> School of Medical Sciences
>> University Walk
>> Bristol BS8 1TD
>>
>> [hidden email]
>>
>>
>>
>> On 26/06/19, 4:22 PM, "Confocal Microscopy List on behalf of Jens Bernhard Bosse" <[hidden email] on behalf of [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.
>>   *****
>>
>>   Dear List,
>>
>>   We are having a problem with field flatness on our Yokogawa-W1-Borealis Unit using Andor 888’s as cameras. We measure a drop of intensity of about 60% from the middle to the sides after a rigid adapter was installed between the stand and the unit.
>>   Did anyone encounter similar problems or does anyone maybe have a service manual?
>>
>>   Thank you!
>>
>>   Jens
>>
> ___________________________________________________________________________
> This e-mail is confidential and is for the addressee only.   Please refer to
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