PSF asymetry

Posted by lechristophe on
URL: http://confocal-microscopy-list.275.s1.nabble.com/PSF-asymetry-tp7583681.html

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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