>Hi Mike,
>
>We have seen a similar problem to this, particularly apparent in DIC 
>transmitted light images.  However, of our two FV1000 systems 
>(located in different rooms), it is only readily apparent on one. 
>This has led us to believe it is a vibration issue, especially as we 
>can cause a more severe, but similar-looking problem by deliberately 
>introducing a source of vibration near to the microscope.  However, 
>if true, we have yet to isolate the cause of the problem vibration.
>
>While I'm here...has anyone properly investigated the effect of the 
>DIC objective prism in confocal fluorescence imaging?  I had always 
>assumed (rightly or wrongly) that it's presence didn't influence the 
>PSF, but last week I was imaging some subresolution beads and found 
>that, particularly on our IX81-based FV1000 confocals, the DIC 
>objective prism had quite a pronounced effect on the psf. 
>Specifically the psf was distorted along a diagonal axis and at the 
>point of focus, the bead appeared significantly larger with the 
>prism in place. The implication of this is that for confocal 
>fluorescence imaging, the resolution of the microscope is reduced 
>when the DIC objective prism is in place.  I've also looked on our 
>Zeiss Axiovert 200 and Nikon TE-2000 based systems which employ a 
>slightly different method of DIC and there the effect is much less 
>pronounced although noticeable.
>
>Simon


Hi Simon,

You raise an important point. As I understand it, it all depends on 
the polarization direction of the laser light. If the pol direction 
is as it should be for DIC operation (i.e., at 45 deg to the tow axes 
of the Wollaston prism) then, yes, the light should form two spots 
displaced by an amount that depends on the prism but will be close to 
the resolution limit of the optics. The direction of the displacement 
should be the same as that of the "shadows" in the DIC image.

However, there are some wrinkles. One is that, because the two beams 
have different polarizations, they may not both excite fluorescence 
in the same way. In particular, if you were looking at the signal 
from a single molecule, it might be lined up to be excited by one 
beam but not the other, (in which case the PSF would be round but 
perhaps slightly displaced) or it might be somewhat excited by both 
(in which case it would be more or less distorted at the DIC angle).

Even on large objects such as beads, where pol isn't important, the 
relative ellipticity will depend on the extent to which the pol of 
the laser is in fact correctly oriented with the Wollastons. (This 
can usually be checked by introducing a polarizer into the microscope 
tube.) If the laser sends in light polarized in the only the ordinary 
(or only the extraordinary) direction of the prism, then the only one 
beam will be produced and the result will again be round.

I don't know why you found that the effect was more apparent on the 
Olympus than on the Zeiss, but as you know, the DIC prisms are cut 
differently for every objective and each one is always a compromise 
between parameters that will produce good contrast versus uniformity 
over a large field of view and I assume that this will affect the 
"real-space" displacement between the two beams. I suppose that the 
manufacturers could tell you the parameters that they use.

Glad that you brought it up.

Cheers,

Jim P.
-- 
               ****************************************
Prof. James B. Pawley,               		   Ph.  608-263-3147 
Room 223, Zoology Research Building,                         FAX  608-262-9083
250 N. Mills St., Madison, WI, 53706  [log in to unmask]
"A scientist is not one who can answer questions but one who can
question answers."  Theodore Schick Jr., Skeptical Enquirer, 21-2:39