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Big difference. The oil negates any coverslip tilt. This brings us back to
the possibility that there is something misaligned in your microscope.
Jiggle anything you have access to, like filters, and if you can, check the
position of your tube lens. Also see if/how much the problem changes with
different objectives.

Craig
On 2013-09-13 5:47 PM, "Serkan Berk" <[log in to unmask]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I realized that the uploaded image in my first message was taken with 100x
> oil objective not with 63x water obj.
>
> Image taken with 63x water is here,
>
> https://www.dropbox.com/s/9g0swtwgueorere/63x%20water%20obj.%205s%2025%25%20110nm.tif
>
> I also uploaded z-stack with water objective,
>
> https://www.dropbox.com/s/49xl9csoxxzijov/110nm%2063x%20water%20obj.%200.173%20axiocam%202x%201s%2025nm%20step%20-5um%20to%20%2B5um%20z-stack_MMStack.ome.tif
>
>
>
> Changing the collar setting to a greater value makes nice looking airy disk
> when the focal plane is above the particle, while setting collar to a lower
> value makes nice airy disk when focal plane is set below the particle. But
> in both conditions PSF in the axial plane becomes more asymmetric than the
> PSF at correct collar setting. Also intensity is highest at correct collar
> position.
>
> Thanks
> Serkan
>