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Hi 

The concentric circles sound like the Newton rings. Are you sure they are fluorescent, not caused by laser light? Can you check your filters, make sure they remove excitation light. Good luck.

De

De Chen, Ph.D. (Contractor)   
Scientist I                                                  
Optical Microscopy and Analysis Laboratory
http://atp.ncifcrf.gov                                 
SAIC-Frederick, Inc.                            
National Cancer Institute at Frederick 
Post Office Box B – see note below   
Frederick, MD 21702                         
Phone: 301-846-7671
Fax: 301-846-7672
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________________________________________
From: Sandrine Pouvreau [[log in to unmask]]
Sent: Thursday, June 09, 2011 10:22 AM
To: [log in to unmask]
Subject: Troubleshooting: large fluorescent circle on an inverted microscope.

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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