bars across field of view

[sandra schulze]

Hi there...

I wonder if anyone out there can explain an image problem I am having
with my fluorescence microscopy. I am currently using a Zeiss Axioscop
but I have seen the same thing on a Nikon some years back, so think
this might be independent of brand.

1. Dark bars, like a fuzzy grid, appear across the field of view.
2. They are only visible when there is broad fluorescence throughout
the field of view. Ie., I do not see them in the DAPI channel b/c the
image is mostly black space with lots of bright blue spots. But I see
it in the GFP channel if I am looking at ALOT of fluorescence
throughout.
3. So far, I have only seen this on the GFP channel, but then that is
the only channel I have that showed that broad fluorescence (the scope
is new - the red and blue have been more defined localizations based
on the antibodies/dyes). But I suspect that if I had lots of rhodamine
staining, I would see it on the red channel also (it was with
rhodamine I remember seeing it on the Nikon).
4. I see it through the 40X and 60X lenses, but not the 10X (and I
haven't got a 100X yet).
5. It does not move with the slide, so it isn't on the cover slip. In
any case, it appears over multiple samples.
6. I have examined the filter cubes and they are spotless.
7. I have cleaned all the lenses, thinking there may be a fingerprint.
But the bars are still there.
8. The light source for this scope is LED (Zeiss's colibri system, not
an arc lamp) and as per the advice of the tech I have rotated the
modules, no change in the orientation of the bars. (Anyway the Nikon
was not LED, it was Hg.)

I would like to attach an image but I think that might not be allowed,
so if someone knows what I am talking about, please could you contact
me and I will send an image. I would like to get this sorted out while
the scope is still under warranty (couple of months left...)

Many thanks,

Sandra Schulze
Assistant Professor
Dept. Biology
Western Washington University
Bellingham WA 98225

[Michael Rule]

Hi Sandra, 

Some suggestions from a microscopy guru friend of mine.... if you want to send an image or two, feel free and I'll play intermediary!

If it’s forming a grid, it doesn’t sound like a dirt/dust problem. First, check to make sure that there isn’t some type of polarizing filter engaged in the lightpath. From your description, I would check the camera next.

 

Can you see these lines through the eyepieces and the camera, or just the camera?

 

If you don’t see it through the eyepieces, check to see if it is some sort of camera issue. If you can, loosen the camera slightly and gently rotate it back and forth 15-30º or so while observing a live image of your sample on the computer. The image of your sample should rotate on the screen as you rotate the camera. What does the “fuzzy grid” do? If the grid rotates with the sample, then the problem is in the scope. If the grid stays fixed in the image while the sample image rotates, then the problem is in the camera (or software).

 

Maybe this will help narrow things down…..

 

----
Michael Rule
ctac.mbi.ufl.edu

352.392.0555
A large number of electrons were terribly inconvenienced in the sending of this message.

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