Dear Simon,

The DIC prism degrades the image quite substantially on our Leica SP2.  I
understood this to be because the fluorescence is "split", but maybe the
laser excitation is affected also.  It has a quite noticable
fuzzing/doubling effect on the final image.  I always recommend that for DIC
overlays, people take two images - fluorescence first, DIC plus fluorescence
second.  Have not looked at the psf, though.

cheers,
Rosemary

Rosemary White
CSIRO Plant Industry
GPO Box 1600
Canberra, ACT 2601
Australia

ph 61 2 6246 5475
fx 61 2 6246 5334
 


On 8/10/09 12:25 AM, "simon walker (BI)" <[log in to unmask]> wrote:

> 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
> 
> 
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[log in to unmask]] On
> Behalf Of MODEL, MICHAEL
> Sent: 07 October 2009 12:28
> To: [log in to unmask]
> Subject: exorcising spirits from Fluoview
> 
> I apologize if this is a second message to the list, I think the first one
> didn't go through
>  
> We are having a bizzare scanning problem. Straight vertical lines in an object
> become slightly zigzagged with a period of up to 6-7 scan lines, and there
> also may be some oscillation in the intensity. The period and the magnitude of
> this periodic noise depends on the scan speed and the scan size. So far we
> (with the help of an Olympus engineer) have established that:
>  
> 1. It doesn't seem to be the scanner controller or the galvo mechanism
> 2. It does not seem to be the electric power in the building
> 3. It is not a mechanical vibration
> 4. It is not a computer
> 5. It is not the cables
> 6. It is not a 60 Hz noise
>  
> Sometimes connecting the scanner controller to the outlet through a long
> extension cord seemed to help which may suggest a problem with grounding, but
> as soon as we concluded that, the trick stopped working. The trouble could not
> be reproduced at the Olympus testing lab.
>  
> Has anyone experienced anything similar and successfully resolved the problem?
>  
> Many thanks in advance!
>  
> Mike Model