Shift in GFP-spectrum

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Sven Terclavers Sven Terclavers
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Shift in GFP-spectrum

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear list,

 

I have been performing spectral analysis experiments on pig-hearts injected with GFP-transfected stemcells with the Zeiss LSM510 META to define whether GFP was present or not. 1 to 3 days after injection, a spectral curve similar to GFP with a clear peak around 510 nm can be observed, though when scanning hearts 6 weeks after injection (with the same settings of microscope, laser and detectors), I only observe a broader spectral curve with a peak around 540nm, thoug with almost the same intensity.  Since it is very crucial for the project to know whether the stemcells are still present or not after these 6 weeks, I want to make sure that the spectrum I observe is (not) GFP.

 

So the question: is it possible GFP has a spectral shift of about 30nm, and gets a broader spectral curve, due to e.g. change in pH, presence of other proteines or whatever other influences? In addition, if so, is there any good reference available to this phenomenon?

 

Thanks in advance,

 

Sven Terclavers

 



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Michael Weber-4 Michael Weber-4
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Re: Shift in GFP-spectrum

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hi Sven,

did you already measure non-GFP hearts as a control? Maybe one of your
curves represents autofluorescence.

cheers,
Michael


Sven Terclavers wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear list,
>
>  
>
> I have been performing spectral analysis experiments on pig-hearts
> injected with GFP-transfected stemcells with the Zeiss LSM510 META to
> define whether GFP was present or not. 1 to 3 days after injection, a
> spectral curve similar to GFP with a clear peak around 510 nm can be
> observed, though when scanning hearts 6 weeks after injection (with the
> same settings of microscope, laser and detectors), I only observe a
> broader spectral curve with a peak around 540nm, thoug with almost the
> same intensity.  Since it is very crucial for the project to know
> whether the stemcells are still present or not after these 6 weeks, I
> want to make sure that the spectrum I observe is (not) GFP.
>
>  
>
> So the question: is it possible GFP has a spectral shift of about 30nm,
> and gets a broader spectral curve, due to e.g. change in pH, presence of
> other proteines or whatever other influences? In addition, if so, is
> there any good reference available to this phenomenon?
>
>  
>
> Thanks in advance,
>
>  
>
> Sven Terclavers
>
>  
>
>
>
> Disclaimer: http://www.kuleuven.be/cwis/email_disclaimer.htm for more
> information.
Sven Terclavers Sven Terclavers
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Re: Shift in GFP-spectrum

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Michael,

That was the first thought, however, even though autofluorescence is present
and the curve is vaguely similar (though not exactly), the intensity is
dissimilar (lower). The observed curve is on the other hand also not
observed in the 1-3 days after injection sections...here the
autofluorescence curve is even more different.
Best regards,

Sven

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Michael Weber
Sent: Thursday, August 16, 2007 2:35 PM
To: [hidden email]
Subject: Re: [CONFOCAL] Shift in GFP-spectrum

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hi Sven,

did you already measure non-GFP hearts as a control? Maybe one of your
curves represents autofluorescence.

cheers,
Michael


Sven Terclavers wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear list,
>
>  
>
> I have been performing spectral analysis experiments on pig-hearts
> injected with GFP-transfected stemcells with the Zeiss LSM510 META to
> define whether GFP was present or not. 1 to 3 days after injection, a
> spectral curve similar to GFP with a clear peak around 510 nm can be
> observed, though when scanning hearts 6 weeks after injection (with the
> same settings of microscope, laser and detectors), I only observe a
> broader spectral curve with a peak around 540nm, thoug with almost the
> same intensity.  Since it is very crucial for the project to know
> whether the stemcells are still present or not after these 6 weeks, I
> want to make sure that the spectrum I observe is (not) GFP.
>
>  
>
> So the question: is it possible GFP has a spectral shift of about 30nm,
> and gets a broader spectral curve, due to e.g. change in pH, presence of
> other proteines or whatever other influences? In addition, if so, is
> there any good reference available to this phenomenon?
>
>  
>
> Thanks in advance,
>
>  
>
> Sven Terclavers
>
>  
>
>
>
> Disclaimer: http://www.kuleuven.be/cwis/email_disclaimer.htm for more
> information.


Disclaimer: http://www.kuleuven.be/cwis/email_disclaimer.htm
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