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Do you have any DIC components in place behind the objective?  They will reflect, and tend to reflect more with shorter wavelengths.

Regards,
Glen
Glen MacDonald
Core for Communication Research
Virginia Merrill Bloedel Hearing Research Center
Box 357923
University of Washington
Seattle, WA 98195-7923  USA
(206) 616-4156
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On Jun 9, 2011, at 8:55 AM, POUVREAU SANDRINE wrote:

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> 
> Hi and thanks for your input. Yes, I thought about this and tried to move the detection window away from the laser wavelength, but it did not solve the problem.
> 
> 
> ________________________________________
> De : Confocal Microscopy List [[log in to unmask]] de la part de Anda Cornea [[log in to unmask]]
> Date d'envoi : jeudi 9 juin 2011 17:53
> À : [log in to unmask]
> Objet : Re: Troubleshooting: large fluorescent circle on an inverted microscope.
> 
> My guess is that you are imaging light reflected off the glass.  Can
> you move the detection wavelength window further to the right, away
> from the excitation line?
> 
> Sent from my iPhone
> 
> On Jun 9, 2011, at 7:22 AM, "Sandrine Pouvreau" <[log in to unmask]
>> 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 thi
>> ck
>> 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