FSC vs Time

Gerhard Nebe-von-Caron Gerhard.Nebe-von-Caron at Unilever.com
Wed Aug 5 04:30:23 EST 1998



          Assuming that you got the axis labels the wrong way round it 
          looks that if you change to hi flow you get initial loss of 
          data because your signal goes below trigger level, assuming 
          you trigger on FALS. Try triggering on fluorescence which is 
          usually ample.
          Secondly ensure you have your beads dissolved in sheath to 
          avoid a mismatch in refractive index.  Using distilled water 
          can act like a lens inside the system.  Whilst this can be 
          used for optimising the alignment, one could actually use 
          the effect to modify the system response of a flow 
          cytometer...
          The widening of the line with increased flow rate should 
          always be to the lower signal intensity as initially the 
          beads should run through the centre of the laser beam, such 
          receive the maximum number of photons.  When you increase 
          the flow more beads will go away from the beam centre and 
          therefore receive less light, thus giving lower scatter.  If 
          the signal increases it indicates that with the higher flow 
          they can suddenly come to the beam centre they did not reach 
          before. ! Signal increase can also occur for light scatter 
          if background particles (gunk or bugs) suddenly come through 
          coincident with your bead or, if you look at fluorescence, 
          free flourochrome surrounds your particle of interest.  
          
          Happy flowing
          Gerhard
          


______________________________ Reply Separator _________________________________
Subject: FSC vs Time
Author:  william_nostrom at med.unc.edu at INTERNET
Date:    05/08/1998 03:37


Hi all

I have a question. Has anyone looked at FSC (or any parameter) vs Time
with beads as a QC/alignment test? A quick  explanation: If you run
beads on a  FSC vs Time dotplot on low fluid control, you should get a
narrow but straight line. If you flip the fluid control switch to HI,
the line should widen, but
stay on the same straight path:

TIME|| xxxXXXXxxxxXXXxxxx
                     FSC

I was shown this little trick a few weeks ago for our sorter, and
applied it to our FACScans. We have one Scan that has been giving us
troubles with CV's and one that is ok. The one that is giving us the
problems with CV's looks fine for FSC vs time, but the one that is ok
gives me a strange pattern:

TIME|| xxxx        xxxxx        xxxxxx
    ||          XXX         XXX
                        FSC

So, I have 2 questions. 1st, what is the benefit of running this test? I
am assuming that it is an indication of proper stream placement in the
laser. If the narrow core stream is where it should be, then the wider
one should be right too. Right? 2nd question. If this second pattern is
telling me my laser isn't right, why are my CV's good? Our BD rep was
just here and says it is fine.  Oh, another thing. I only see this on
FSC vs Time. When I look at SSC or FL1, everything looks ok. Any
suggestions?
TIA,
Bill Nostrom



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