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Christophe, 

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?

Cheers,

John Oreopoulos


On 2015-04-22, at 5:30 AM, Christophe Leterrier wrote:

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> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
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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 <
> [log in to unmask]> 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
>>