[Cytometry] Ghost population?

WEHICytometry facs_copy at wehi.EDU.AU
Thu Feb 5 23:04:46 EST 2009


Rachael,

Fluorescence compensation will press your negatives onto the axis but  
there is another effect we've seen on MoFlo that sounds more like  
what you describe.  Check your detector pulse waveform for the  
offending PMT.  If it has a ripple between cells, that will split  
your negative population.  This ripple may be coming from a  
maladjusted fluorescence obscuration bar, but the PMT power and/or  
signal cables can also pick up AC waveforms from the air.  We have  
been able to reduce the latter by running the cables from the PMTs  
straight down between the detector modules to the preamp box (rather  
than have them stick up like antennae).

And yes, what ever the cause of the cells sticking to the axis, your  
gates should go all the way down.

Frank Battye.

On 05/02/2009, at 3:28 AM, Rachael Walker wrote:

> I am now the proud owner of a 50mW 561nm laser for my MoFlo (I change
> over my 561 and red diode depending on sort) and have been using it a
> lot for RFP and it seems to be working well. However, I see a  
> population
> of cells against the axis by my negatives. I have have heard of a  
> ghost
> population appearing on a red channel, am I seeing the same thing  
> on my
> RFP channel? Is there anyway of getting rid of it and also what is a
> ghost population? I know I will be asked about it by my punters and I
> need to explain it in a scientific way and not by the explanation I am
> using a the moment which is, its a ghost population, it doesn't really
> matter, they really are negatives and its there because there isn't
> enough light getting through.
>
> I see it using  590/20, 610/20, 613/20  filters, on negative cells as
> well as positive populations.  I do also see it with my red diode
> laser.  When gating cells do I include the ghost population?   
> Should my
> gates go all the way to the axis?

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