[Fluorescent-proteins] bars across field of view

Michael Rule via fluorpro%40net.bio.net (by mikerule from ufl.edu)
Fri Aug 20 17:41:32 EST 2010


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.




On Aug 19, 2010, at 4:51 PM, sandra schulze wrote:

> 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
> 
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