[Cytometry] Ghost population?

Rui Gardner ruig at igc.gulbenkian.pt
Thu Feb 5 17:36:22 EST 2009

Hi Rachael,

We've been using a 50mW 561nm laser in our moflo for quite some time 
now, and never observed this "ghost" population you refer to. We 
actually have the 561 laser coupled through a fiber optical cable so we 
can switch the laser from one side to the other, and so we've even used 
the 561 and red laser together and still haven't seen this population.  
Are you sure these are cells? What kind of PMT are you using and what 
are the voltages you're setting it to?

I do recall once seeing an extra population arising when I was cranking 
up the voltage of one of my Hamamatsu H957-15 PMT's to detect PE-Cy7 
(with the 488nm), but then I ran the negative sample again and realized 
it was also present. Later I read this is related to large noise due to 
excessive amplification of the PMT (I set the voltage to over 900), but 
it didn't explain why an extra population appears (and I must apologize 
for my lack of knowledge in this matter). Could this be what you are seeing?

Rui Gardner
Flow Cytometry Lab Manager
Instituto Gulbenkian de Ciência
Apart. 14, PT-2781-901 Oeiras
Email: ruig at igc.gulbenkian.pt

Rachael Walker wrote:
> Dear All
> 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?
> Many thanks
> Rachael

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