Re: Image correction help!

Posted by Wolfgang Schechinger on May 08, 2007; 10:34pm
URL: http://imagej.273.s1.nabble.com/Please-Divide-an-Image-with-ImageJ-tp3699513p3699520.html

Hi Don,

can you define a structure which is only labeled by one dye? Then you could measure the contribution of one dye to the crosstalk and determine the constant you have to multiply one image before subtracting it from the other. Maybe you have to make an experiment where you omit one of the dyes in question.

Just some thoughts,

Wo


-------- Original-Nachricht --------
Datum: Tue, 8 May 2007 12:29:36 -0400
Von: "Liu, Dongfang (NIH/NIAID) [F]" <[hidden email]>
An: [hidden email]
Betreff: Image correction help!

> Dear all,
>
> Recently, I came across a problem with alexa568 and alexa647 on our TIRF
> system. I have to label my two proteins using the two different dyes
> (alexa568 and alexa647)simultaneously(because I have to use four dyes in one
> chamber, alxa488, alexa555, alexa568 and alexa647).
>
> Problem: When I used 568nm laser to excite the alexa568 dyes on lipid
> bilayer(the emission filter is LP665), I got the fluorescence of alexa647 in
> alexa568 emission channel(because 568 laser can partially-10% excite the
> alexa567 laser.).
>
> The protocol for acquiring image alexa568 and alexa647:
> 1, Acquire the alxa568 and alxa647 image in sequence.
> 2, Excitation laser for alexa568 is 568nm, emission for alexa568 is LP665
>    Excitation laser for alexa568 is 647nm, emission for alexa647 is LP665
>
> >From the spectrum properties, we can know the fluorescence of Alexa647
> can not breakthrough into alexa568 channel(even I used the same emission
> filter to acquiring the fluorescence of alexa568 and alexa647, because 647nm
> laser cannot excite the alexa568 dye)
>
> What I want to do: I want to remove the fluorescence of alexa647 from
> alexa568 channel. How to do it? Any comments would be greatly appreciated!
>
> Thank you very much! Have a nice day!
>
> Best regards,
>
> Don

--
"Feel free" - 10 GB Mailbox, 100 FreeSMS/Monat ...
Jetzt GMX TopMail testen: http://www.gmx.net/de/go/topmail