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The first part of this post is primarily in response to SEK-Claude Cantin
recent post on high speed sorting. As much of his post was obviously
directed toward me, I'm compelled to respond. (Round 2) Just to clarify,
not to antagonize. Well, maybe a little. Downright HOT.
I didn't intend to suggest coincident events were not a source of sort
impurities. I'm sure Claude saw a big improvement in sort purity by
increasing cell concentration and decreasing sample pressure. There are
probably many people on the net that will benefit from his advice. My
only point was that at some point the speed of the electronics become the
limiting factor in sort purity. I'm assumimg Claude's sorter is setup
perfectly in terms of drop-delay, etc.. But given the cell concentrations
and sorting conditions he described and getting 1.5% impurities, it's
obvious something more than coincident events are responsible.
Below are the results of a quick experiment I devised to evaluate the
Cicero electronics from Cytomation on a FACScan. The data represent
trigger rates (events/sec) for PI stained nuclei. Dilute and concentrated
samples were run on high and low sample pressure before and after the
Cicero electronics were attached to the FACScan. Hi:Lo = Ratio of rates
DILUTE ! CONCENTRATED
Low P. High P. Hi:Lo ! Low P. High P. Hi:Lo
LYSYS II 250 930 3.72 ! 1200 3500 2.92
!
Cicero 260 1080 4.15 ! 1360 5120 3.76
This obviously not a rigorous, scientific comparison. A friend of mine
has a Cicero on an EPICS 753 and can monitor the trigger rate through
the MDADS and Cicero simultaneously. He gets similar results.
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- From Kris Weber's data:
I wanted to confirm that the data rate would be proportionate to the
concentration of beads in a sample. I started with a bead sample of 1.3 x 10(8)
and did 1:1 dilutions down to 2.08 x 10(6). The chart shows what happened.
Concentration Actual data rate Theoretical data rate
1.3 x 10(8) ND 32,000
6.5 x 10(7) 4000 16,000
3.25 x 10(7) ND 8,000
1.67 x 10(7) 2400 4,000
8.35 x 10(6) 1800 2,000
4.17 x 10(6) 1000 1,000
2.08 x 10(6) 500 500
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ARE WE SEEING A TREND YET? As I said in my original post, at high rates
the electronics can't keep up with the number of cells flowing through the
system. This is why it's virtually impossible to obtain >99% purity for a
population that's less than 5% at rates exceeding 4,000 events per second
on most commercially available instruments.
I don't even want to talk about yeild. Then my blood will really start
to boil. Thanks to Eric Van Buren for catching my error in definition.
Getting back to Claude:
> Finally, loss of 'sortable' events to the waste tank probably has more
>to do with setup (oscilator frequency vs sheath pressure, drop delay
>measurement, phase adjustment, drop delay offset ("QUESTION
>AUTHORITY"...who says the offset IS correct for your setup?), and
>unfortunately once again, coincidences.
As a matter of fact my drop delay offset is off, by 1/2 drop - I have to
correct for it. If you consider me an authority I'll take that as a
compliment. What I had in mind when I wrote the "Question Authority" was
to question everything. I wrote that in graduate school after spending
months trying to reproduce an "authority's" results and finally realizing
the "authority" was flat wrong. I would question the authority that says:
>Dead time window is not a factor because 20 - 40 thousand events
>per second can be processed with a dead time window of 15 to 45
>microseconds (standard Facstar setup).
How does that jive with the data above?
OK, I admit it, my response to your initial post could be viewed as
somewhat sarcastic. It wasn't meant to ridicule. It was, in part, due
to my frustration with the instrumentation. Yes, you can tell when
someone is yelling on the Net.
And by the way, the reason you need a doublet discriminator for DNA
analysis is that on FACS systems, you are using (I hope) the fluorescence
"Area" parameter. This is BD jargon for integrated fluorescence - the
fluorescence signal is run through an integrating amp. Integrating
amplifiers are notorious for identifying doublets as singlets.
I'm really not a flow-jock, I only play one on the internet.
In real life I'm a biologist.
tom d.
"Never, for the sake of peace and quiet deny your own experience
or convictions"
Dag Hammarskjold
-- ============================================================================== Thomas Delohery | Internet: t-delohery@ski.mskcc.org Manager, Flow Cytometry Core Facility | Phone: (212) 639-8729 Memorial Sloan-Kettering Cancer Center | Fax: (212) 794-4019 ==============================================================================
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