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Hello Marc: We have realized some blind unmixing, almost always we use this function, you have to select the number of expected channels in order for this to work better, sometimes a change in the width of the spectral capture also helps, so we used a combination of opening the confocal aperture with a shorter step size, 5nm or 2nm depending on the distance between peaks of the dyes involved, and a band width at 7nm or less, this usually compensates when we see a low emission on the sample.  I hope this helps Gabriel OH
 > Date: Wed, 12 Oct 2011 10:55:21 -0400
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> Subject: blind unmixing
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> HI,
> 
> We are experiencing some difficulties doing blind unmixing with the Olympus
> FV100 on double-orange beads. 
> 
> Have someone been successful with this technique ? If not, we could also
> enter the spectral data of the 2 fluorophores to do normal unmixing, but
> this too seems problematic. Any info or protocol would be enormously
> appreciated. 
> 
> Thank you very much in advance.
> 
> Marc
> 
>  
> 
>  
> 
> Marc Thibault, PhD
> 
> Chemical Engineering Department  
> 
> Ecole Polytechnique of Montreal
> 
> tel: (514) 340-4711 ext.3968
> 
>  
> 
>