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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:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> 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,
> 
> [log in to unmask]
> 
> Sent from my iPhone
> 
>> On Jun 26, 2019, at 4:40 PM, Mark Cannell <[log in to unmask]> 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
>> 
>> [log in to unmask]
>> 
>> 
>> 
>> On 26/06/19, 4:22 PM, "Confocal Microscopy List on behalf of Jens Bernhard Bosse" <[log in to unmask] on behalf of [log in to unmask]> 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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