I recently had this same problem with one objective on a new confocal installation here. After acquiring a XYZ stack documenting the lateral shift of the PSF I rotated the objective 90 degrees and acquired another stack. The lateral shift rotates the same as the objective rotates therefore the defect is in the objective. The manufacturer is replacing the objective.
Cheers, Michael
Michael Weis
Supervisor, Superviser,
Microscopy Facility Installation de microscopie
Science and Technology Branch Direction générale des sciences et de la technologie
Pacific Agri-Food Research Centre Centre de recherches agroalimentaire du Pacifique
P.O. Box 5000, 4200 Highway 97 Boîte postale 5000, 4200 Autoroute 97
Summerland, BC, V0H 1Z0, Canada Summerland, CB, V0H 1Z0, Canada
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Christophe Leterrier
Sent: April-21-15 1:10 AM
To: [log in to unmask]
Subject: PSF asymetry
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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
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