FocalCheck test slide

[Paul Herzmark]

Guy Cox wrote  to CONFOCALMICROS.4:25 AM (3 hours ago)

No, no, it IS the problem!  As Mike Ignatius explained, MP put the beads on the slide, not the coverslip, then add mountant and then the coverslip.  If you are using a #1.5 coverslip you need to put the beads directly on the coverslip.  With the beads on the slide the extra thickness of the mountant needs to be corrected for by using a thinner coverslip - #1 or #0 - which must be found by trial and error.  (But since I guess they are using very reproducible conditions they only need to do the test once).  At least it seems they have realized they have a problem.
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Invitrogen has known that they make their bead test slides wrong since at least July 2005. I copy below an email exchange I had with the company. The conversation started on the Confocal List Serve.

I edited the exchange for clarity.  

Paul Herzmark
Department of Molecular and Cell Biology
University of California, Berkeley

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> From: Confocal Microscopy List July 19, 2005 10:57 AM

Why are the beads not mounted with a 1.5 coverslip (0.17mm)? Microscopes are designed for that thickness and there will be significant spherical aberration with a #1 coverslip (especially with a high dry objective).
Paul Herzmark



On 7/19/05, Clements, Ian <[hidden email]> wrote:

Thank you for your comments regarding the coverslip thickness issue.
 
We were aware that certain high-NA lenses may have issues with the choice of coverslip thickness.  Due to the slightly higher RI of the mounting cement (1.52-1.53) the thickness of the coverslip is not as critical for standard oil immersion systems where the RIs of the lenses, immersion media and sample are near matches.  The whole sample is in essence a single piece of glass.
 
Unfortunately the situation you describe for a High-Dry lens is very likely true but one we have no ready ability to test and evaluate.  Replacing the coverslip on future batches of product is relatively straightforward and if you are interested I would welcome your input on the difference if you'd be willing to evaluate such products.

I'll can see about getting another batch made using the 1.5 coverslips and I can send you one of each for comparison if you are willing to share the results.
 
Ian Clements
Invitrogen Corp

From: Paul Herzmark
July 19, 2005

Hi Ian,
I also do not have a high magnification air objective lens to evaluate the aberration. But why test it? It is a known phenomenon that would be avoided with the 1.5 coverslip.

And thanks for replying to me!
Paul

Jul 19, 2005

Thanks Paul.
I'll still probably try to have someone do a side by side comparison as I'm really curious to have some on hand image data that could be used for testing.  After all this is one of the things we are trying to get at with these slides so the more data we have the better.  Perhaps if Zeiss or someone is interested they'll loan us some lenses to play with.

Ian Clements
Invitrogen Corp