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

Posted by Guy Cox on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Coverslip-thickness-and-correction-collar-tp591122p591124.html

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But ... but .....

 

(a) this will be nowhere near accurate enough

(b) setting the ring manually once you know what to

look for takes only a minute or two

 

If you are paying more than $8 per minute for confocal

time you are being seriously overcharged!

 

                                                              Guy

 

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
    http://www.guycox.com/optical.htm
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Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
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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

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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. 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.



At 08:25 AM 12/6/2007, you wrote:

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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]
-------------------------------------------------

 

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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