Troubleshooting: large fluorescent circle on an inverted microscope.

[POUVREAU SANDRINE]

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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

[John Oreopoulos]

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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:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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

[Anda Cornea]

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" <[hidden email]
 > wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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

[POUVREAU SANDRINE]

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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 [[hidden email]] de la part de Anda Cornea [[hidden email]]
Date d'envoi : jeudi 9 juin 2011 17:53
À : [hidden email]
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" <[hidden email]
 > wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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

[Glen MacDonald-2]

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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
[phone]
[hidden email]



On Jun 9, 2011, at 8:55 AM, POUVREAU SANDRINE wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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 [[hidden email]] de la part de Anda Cornea [[hidden email]]
> Date d'envoi : jeudi 9 juin 2011 17:53
> À : [hidden email]
> 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" <[hidden email]
>> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> 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

[Chen, De (NIH/NCI) [C]]

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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                                 
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National Cancer Institute at Frederick
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________________________________________
From: Sandrine Pouvreau [[hidden email]]
Sent: Thursday, June 09, 2011 10:22 AM
To: [hidden email]
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

[Andreas Bruckbauer]

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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 <[hidden email]>
To: [hidden email]
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


 

[POUVREAU SANDRINE]

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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 [[hidden email]] de la part de Andreas Bruckbauer [[hidden email]]
Date d'envoi : jeudi 9 juin 2011 23:34
À : [hidden email]
Objet : Re: Troubleshooting: large fluorescent circle on an inverted microscope.

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

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 <[hidden email]>
To: [hidden email]
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