Re: Coverslip thickness and correction collar

Posted by Guy Cox on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Coverslip-thickness-and-correction-collar-tp591122p591133.html

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

If it wasn't 'picky' it wouldn't be doing its job!
Correct adjustment is crucial and without it you'll
be doing worse than using a lower NA lens.  Forget
looking for more accurate cover slips - at this level
sufficiently accurate ones don't exist so all it would
do is give you a closer starting point.

Method 2 is the way to go but the key is not to look
for best image quality (even though this is what you'll
obtain, it's hard to spot).  What you are looking for
is IDENTICAL IMAGES EITHER SIDE OF FOCUS.  If your spot
goes to a circle one side of focus and a spot with a
halo around it on the other side, you have SA.  Adjust
the collar (widefield, visual observation) till the image
looks identical either side of focus.  It will actually
go to a bit of a ring on either side, and this is normal
(a look at a diagram of the psf will show you why).  It's
easiest to do this in fluorescence but if fading is a
problem it can also be done in brightfield.

                                              Guy


Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
    http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
______________________________________________
Phone [phone]     Fax [phone]
Mobile [phone]
______________________________________________
     http://www.guycox.net
-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Teemu Ihalainen
Sent: Friday, 7 December 2007 12:26 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]
-------------------------------------------------

No virus found in this incoming message.
Checked by AVG Free Edition.
Version: 7.5.503 / Virus Database: 269.16.17/1177 - Release Date: 7/12/2007 1:11 PM
 

No virus found in this outgoing message.
Checked by AVG Free Edition.
Version: 7.5.503 / Virus Database: 269.16.17/1177 - Release Date: 7/12/2007 1:11 PM