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What did you use as a mounting media? Do you see the lateral shift when you look through the eyepieces?

> On Apr 21, 2015, at 4:10 AM, Christophe Leterrier <[log in to unmask]> wrote:
> 
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> 
> 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

Eric Marino
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