Re: Coverslip thickness and correction collar ... option Ib

Posted by George McNamara on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Coverslip-thickness-and-correction-collar-tp591122p591126.html

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The only way to know which - if either - was the correct answer is to
stand the coverglass on edge, and measure that. Did you do this?

I did not mention this with respect to the dishes, because that
measurement would require breaking or unglueing the glass.

Jim P - thanks, thanks, and thanks again, for your point about where
the lenses are corrected for.



At 08:52 AM 12/10/2007, you wrote:

>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>>
>>From: Confocal Microscopy List
>>[mailto:[hidden email]] On Behalf Of George McNamara
>>Sent: Sunday, 9 December 2007 6:08 AM
>>To: [hidden email]
>>Subject: Re: Coverslip thickness and correction collar ... option Ib
>>
>>Search the CONFOCAL archive at
>>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>>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
>[hidden email]
>
>
>
>
>>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
>>[hidden email]
>>[hidden email]
>>305-243-8436 office
>>http://home.earthlink.net/~pubspectra/
>>http://home.earthlink.net/~geomcnamara/
>>http://www.sylvester.org/health_pro/shared_resources/index.asp (see
>>Analytical Imaging Core Facility)
>>
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>
>George McNamara, Ph.D.
>University of Miami, Miller School of Medicine
>Image Core
>Miami, FL 33010
>[hidden email]
>[hidden email]
>305-243-8436 office
>http://home.earthlink.net/~pubspectra/
>http://home.earthlink.net/~geomcnamara/
>http://www.sylvester.org/health_pro/shared_resources/index.asp (see
>Analytical Imaging Core Facility)
>
>