Re: Coverslip thickness and correction collar

Posted by Armstrong, Brian on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Coverslip-thickness-and-correction-collar-tp591122p591134.html

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Well, I think you should start with method I, and then use method II for fine tuning. After all it is the image quality you are after regardless of what numbers on the collar are used.
It is my understanding that Zeiss uses the tube lens for correction whereas Olympus corrects entirely in the objective (see Handbook of Biological Confocal Microscopy, Pawley J). Which begs the question, why are you putting an Olympus lens on your LSM510? Try the Zeiss 63x/1.2W Corr and see if you have the same issues.
Cheers,
 

Brian D Armstrong PhD
Light Microscopy Core Manager
Beckman Research Institute
City of Hope
1450 E Duarte Rd
Duarte, CA 91010
626-359-8111 x62872
http://www.cityofhope.org/SharedResources/LightMicroscopy

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Teemu Ihalainen
Sent: Thursday, December 06, 2007 5:26 AM
To: [hidden email]
Subject: Coverslip thickness and correction collar

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear list,

I have a small question considering coverslip thickness correction when
using water immersion objectives and I was hoping that you could help me
out.

We have bought a water immersion objective from Olympus (UPLSAPO 60x, 1.2
NA) with a correction collar. The objective works nicely in our LSM510,
when imaging living cells it gives good images, the PSF is better compared
to oil immersion objectives, etc. So, everything goes according to the
theory.

There is only one problem. The objecive is very "picky" about the
coverslip thickness (again, like the theory predicts...). We are using
glass bottom dishes from MatTek which have the thickness in range of
160-190um. Is there some simple way to adjust the correction collar when
imaging your sample?

The adjustment is easy to do when you are imaging for example ps-specks
but when you have your real sample, it is trickier. I have thought/found a
couple of ways to do it (below) but Im not sure which is the best and are
there other ways to do it.

I
Measure of the glass thickness before you culture your cells. Then at the
microscope you just adjust the collar accordingly.

II
At the microscope you first try to find a really small detail(s) in your
sample and then adjust the collar for best image quality (image z-stacks
or something like that).

III
Image some cell adhering beads(?) what you can just add to your sample.
Unfortunately I dont know if such beads even exists.

IV
Buy some other dishes with more accurate glass thickness. Is there any?

Any suggestions and tips are warmly welcome.



Best Regards,
Teemu Ihalainen
-------------------------------------------------
M.Sc., Nanoscience
University of Jyväskylä
Department of Biological and Environmental science
Molecular Biology, room B 212.2
Survontie 9 B2, 40500 Jyväskylä, Finland
Tel. [phone]
Mobile [phone]
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