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I suppose it's possible, but highly unlikely that Christophe was given two defective objective lenses here. Christophe, is the angle of asymmetry the same for both objective lenses?

John Oreopoulos


On 2015-04-21, at 11:42 AM, Weis, Michael wrote:

> 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
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> 
> 
> 
> -----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