Posted by
James Pawley on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Coverslip-thickness-and-correction-collar-tp591122p591129.html
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>>
>>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]
Hi all.
The dry, coverslip lens is ONLY corrected for imaging immediately
next to the far side of a coverslip of the specified thickness. The
image of the near side will be poorly corrected, and as you noted, in
the wrong place. How much "the wrong place" depends on how well you
fill the BFP because this determines the effective NA on the
illumination side..
Dry lenses are even more fussy that oil lenses of the same NA about
the thickness of the coverslip.
Dry lenses are even more fussy that oil lenses of the same NA about
the thickness of the coverslip.
Dry lenses are even more fussy that oil lenses of the same NA about
the thickness of the coverslip.
This sad news brought to you with best wishes for the Holiday
Season(translation: the time when we get to correct all the papers)
Cheers,
Jim P.
>
>
>
>>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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--
****************************************
Prof. James B. Pawley, Ph. 608-263-3147
Room 223, Zoology Research Building, FAX 608-262-9083
250 N. Mills St., Madison, WI, 53706
[hidden email]
"A scientist is not one who can answer questions but one who can
question answers." Theodore Schick Jr., Skeptical Enquirer, 21-2:39