Dear All,
I am having a problem with a X60 1.4NA oil immersion objective which I am
using in an Olympus IMT-2 inverted microscope. When I look at calibration
beads (MP 0.17um yellow-green fluorescence - PS-Speck kit) they appear with
a strange halo (I don't know how to call it) casting away from the beads
(this in conventional observation) Something like:
 
           /
         / /
        O
         \ \
           \
 
The lines are very weak, but noticeable. They display the same orientation
disregarding the position of the bead in the field. When defocusing, the
circular halos are not centred, but shifted in the same direction. It looks
like it's a problem with the objective, because it's not noticed with other
objectives, but this may be due to the lower power and brightness of these.
It doesn't change with slide orientation or tilt.
 
Now, when scanned (MRC 600), XZ sections are not perfectly vertical but
slightly deviated (in the same direction in both upper and lower frames)
Also, looking at the amplitudes of the FTs of the confocal images, these
look fairly symmetric on XY but they are tilted on XZ planes.
Older images (not beads but microtubules, which I would assume to also show
the effect if present) do not show any deformation. Their FTs do not show
any such tilt.
 
Does anybody have any ideas of what can be happening?
 
Thanks in advance
 
Carlos