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You might inadvertently be creating a confocal backscatter image of the excitation light . This mode of imaging will sometimes produce circular interference patterns. See example images in this paper:

Paddock, S., Confocal reflection microscopy: The "Other" Confocal mode. Biotechniques, 2002. 32(2): p. 274. 

I have also seen this before with 1-photon laser scanning confocal microscopes when accidentally forgetting to insert the emission filter. It can also produce the negative image you described. Ensure that the correct laser excitation wavelength, dichroic mirror, and emission filters are being used for this green fluorophore. Does this make the problem go away?

Also, is it possible that there is a bubble in your immersion oil? Try wiping the slide and cleaning the objective and carefully reapplying the oil.

John Oreopoulos
Research Assistant
Spectral Applied Research
Richmond Hill, Ontario
Canada
www.spectral.ca


On 2011-06-09, at 10:22 AM, Sandrine Pouvreau wrote:

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> 
> Hi there.
> I am doing confocal imaging on neuron cell cultures, using an inverted confocal 
> microscope (Leica SP5 or SP2). The neurons are grown on a 170 µm thick 
> glass, coated with polyLlysine. For imaging, the slices are transferred in a 
> Ludin chamber, which is filled with a non fluorescent saline solution.  The 
> objective is an oil immersion one, 63x. This is theoretically not ideal for live 
> cells, but I never had troubles with this configuration before. The oil is the 
> Immersion liquid Type F from Leica. (I also tried another type of oil, whose 
> name I don’t remember. Now here is the problem: I see big concentric 
> fluorescent circles which cover my entire acquisition field , looking pretty 
> much like an airy disk, which make the image acquisition impossible.  The 
> circles remain in the same area of the picture when I move in xy. However, 
> their shapes change when I change the focus, with a big stain in the middle 
> when I move the objective down (confocal plane closer to the slide), and 
> going outside the field when I move the objective up. Moving the objective 
> down, I sometimes see the neuron in dark on a sea of fluorescence, like a 
> negative picture.  It seems that this phenomenon occurs when I try to image 
> in green, but not in red (DsRed).  Even worst:  I tried to transfer my prep to a 
> spinning disk system using the same microscope, and then I don’t see these 
> circles. I also don’t see this phenomenon if I look at my coverslip with an 
> upright multi-photon. And last, I tried to put a mounting media on my prep 
> with index matching the oil, and it did not solve to problem.  Has anybody any 
> clue about what is going on???
> Thanks!
> Sandrine