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> From: Confocal Microscopy List [mailto:[log in to unmask]] 
> On Behalf Of George McNamara
> Sent: Sunday, 9 December 2007 6:08 AM
> To: [log in to unmask]
> Subject: Re: Coverslip thickness and correction collar ... option Ib
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> Search the CONFOCAL archive at 
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> option Ib. Measure of the glass thickness before you culture your 
> cells. Then, for this lens, use dishes whose coverglasses are 170 um 
> (or whatever thickness works best for the lens at its current 
> "optimal" setting).
>
> If one in five are the right thickness, than the cost of that dish, 
> for this experiment, has gone from about $2 to $10. Should take less 
> than a minute to measure a dish using a 20x, decent NA, dry lens.


Dear George

On a recent test I did, I discovered something that I had not expected. 
A dry objective would significantly underestimate the thickness of a 
glass coverslip.

For a #1 coverslip, a 20x dry objective would measure it as 98microns 
thick, where as a 63oil objective as 138microns thick.
For a #1.5 coverslip the values were 113microns and 160microns 
respectively.

As I said, I wasn't expecting it because the 20 lens was coverslip 
corrected.

So you have to be very careful when using dry objectives to measure 
anything in Z.

regards

*********************************
Stamatis Pagakis Ph.D.
Biological Imaging Unit
Biomedical Research Foundation, Academy of Athens
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> Compared to the cost of the confocal time (or of an LSM510), or of 
> your time to do manual image analysis, or cost of the analysis 
> computer and image analysis software, the extra cost is trivial. Plus, 
> you can sort the "rejects" and use them with lenses each thickness is 
> optimized for.

> George McNamara, Ph.D.
> University of Miami, Miller School of Medicine
> Image Core
> Miami, FL 33010
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> http://home.earthlink.net/~geomcnamara/
> http://www.sylvester.org/health_pro/shared_resources/index.asp (see 
> Analytical Imaging Core Facility)
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