Re: Coverslip thickness and correction collar

Posted by Michael Weber-4 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Coverslip-thickness-and-correction-collar-tp591122p591135.html

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Hey Teemu,

I tried several ways of adjusting the correction collar, and for me the
results are as follows:

- The numbers written at the collar are not very precise. However,
measuring the thickness of coverslip and then put it to the according
number brings you at least in the range of the correct value.

- The way which brings me the best results is to perform a line Z-stack
over the coverslip, and go for the thinnest representation of it at half
maximum. Big disadvantage: this technique is quite a pain without fast
Z-stage.

- An alternative way is to simply go for the highest intensity while
imaging a sample in XY and using a line profile. To my understanding,
when it comes to refractive index missmatches, you lose light due to
refraction. If it matches better, you get more light back into the
objective, thus the best correction collar position should give you the
highest intensity.


I would appreciate if somebody could comment on that.

cheers,
Michael



Teemu Ihalainen wrote:

> 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
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