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Serkan,

That doesn't quite look like coma to me. Where in the field of view was this image taken? By any chance are those beads near the edge of the field of view? Does every bead, even those in the middle of the field of view look like the ones you've shown here? (Note to everyone else who tries to download this image, you have to really stretch the contrast to see the artifact Serkan is talking about. I used ImageJ to do this).

To me, it almost looks like there's something blocking part of the back-aperture of the objective since you see the same "bite" missing out of all the bead Airy disks, and on the same angle. You might want to check the back of the objective, the nose piece, or any other part of the infinity space of the optical train (any other conjugate plane if you have any other periferal devices between the camera and microscope).

Does the pattern change when you change the correction collar?

Just some ideas,

John Oreopoulos
Staff Scientist
Spectral Applied Research
Richmond Hill, Ontario
Canada
www.spectral.ca


On 2013-09-13, at 2:48 PM, Serkan Berk wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
> 
> Hello,
> 
> I am observing some sort of an aberration (looks like coma) when imaging
> sub-resolution sized fluorescent beads under epifluorescence. I am using a
> Zeiss Axio Observer Z1 microscope with a Zeiss C-Apochromat 63x Water Obj.
> NA=1.2.
> 
> I match the collar correction to the coverslip thickness but the airy disks
> are not complete. I uploaded one of the images here
> (https://www.dropbox.com/s/kst4plbrqmv29hn/100nm%20100x%201.0s.tif).
> 
> Image was taken with a sCMOS Camera (Hamamatsu Orca Flash v4.0) attached to
> the side-port of the microscope. Particles are excited with SpectraX light
> engine from Lumencor through a GFP filter set from Semrock.
> 
> Has anyone come across such problem? Let me know if you have any ideas about
> what might be the possible causes?
> 
> Thanks
> Serkan Berk
> 
> University of Minnesota
> 116 Church St. SE
> Minneapolis, MN 55414