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It was indeed the AOBS. We could see the phenomenon almost 100 nm away from the laser line!
Thanks for the help!
Sandrine


________________________________________
De : Confocal Microscopy List [[log in to unmask]] de la part de Andreas Bruckbauer [[log in to unmask]]
Date d'envoi : jeudi 9 juin 2011 23:34
À : [log in to unmask]
Objet : Re: Troubleshooting: large fluorescent circle on an inverted microscope.

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Does your SP5 or SP2 have an AOBS? If yes, this sophisticated beam splitter might not be working properly and letting laser light through?

best wishes

Andreas










-----Original Message-----
From: Sandrine Pouvreau <[log in to unmask]>
To: [log in to unmask]
Sent: Thu, 9 Jun 2011 15:22
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