http://confocal-microscopy-list.275.s1.nabble.com/PSF-asymetry-tp7583681p7583702.html
Yes, it looks like you've got some severe distortion or field curvature in the lower right corner of that image as well. Your question about the camera alignment is a good one and pretty simple to answer. Does an object that appears centred in the eyepiece field of view centred in the camera field of view as well?
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> Dear all,
>
> Thanks a lot for your advices and hypotheses. I think both 100X objective
> give the same asymetry, so maybe the problem is somewhere else (or it is a
> case of bad luck). I have realigned the laser, but it is not the source of
> the problem since it also happens with epifluorescence illumination.
> Attached is an uncropped image of 512x512 image from the EMCCD field of
> view, taken with epifluorescence illumination:
>
>
https://drive.google.com/file/d/0B_JeGjE7nBHWMUFzZEJ0NDFWS1k/view>
> My impression is that the center of the optical axis is not in the center
> of the field of view (does that makes sense?), so maybe a problem with
> camera alignment?
>
> Christophe
>
> On Tue, Apr 21, 2015 at 10:10 AM, Christophe Leterrier <
>
[hidden email]> wrote:
>
>> Dear microscopists,
>>
>> I am using a very nice TIRF microscope with a 100X, 1.49 NA objective. I
>> had the impression that there was a slight lateral shift when defocusing up
>> and down, so I checked the PSF with 100 nm beads on a HR #1.5 coverslip.
>> What appears on the attached image (three planes taken at -1, 0 and +1 um)
>> is that the PSF is not rotationnally symetric, i.e.more intense on the
>> left-bottom side :
>>
>>
https://drive.google.com/file/d/0B_JeGjE7nBHWWFViM0pwSy0xR3M/view>>
>> This asymetry is quite constant over the field of view (it is not radial
>> relative to the center of the field). It does not depend on the
>> illumination (it is the same under azimutal laser, TIRF laser,
>> epifluorescence lamp). It does not depend on the filter cube used. Finally
>> (and this is what surprises me the most), I got another brand new 100X,
>> 1.49 objective for testing and it still shows up (the attached image is
>> taken with the new objective).
>>
>> Do you have an idea if what could be wrong, and how to correct it? Could
>> it be caused by an internal lens? By the sample used?
>>
>> Thanks for your help,
>>
>> Christophe
>>