>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
 
I think we need to know first of all what is the medium the beads
are in.  If there is a refractive index mismatch _and_ the cover-slip
is slightly tilted it could give this sort of effect.  If not it
could be damage to the lens
 
                                        Guy