[Cytometry] Scaling problem with FACSDiva

Ina Laura Pieper inalaurapieper at gmail.com
Thu Feb 6 12:14:46 EST 2014


I've been doing what Chris and Mario describe on my Aria I in setting the
PMTs so that positives are on the plot, and not really worrying about the
negatives since it is easy to visualize them using the biexponential axis.
However, I now have a new Navios and this approach doesn't work! What you
see on the plot seems to be what you get - if the data is falling off the
axis on the left then it's not recorded. Is this a correct observation? Has
anyone else experienced this? What can I do on the Navios to get the same
"BD experience"?

Cheers
Ina Laura
On 6 Feb 2014 02:31, "Mario Roederer" <roederer at drmr.com> wrote:

> Dave Dunaway wrote:
>
> > I find this to be dubious advice unless all you are interested in is
> crunching numbers.
>
> But, in fact, that's ALL we should be interested in.  Graphs are for
> illustration purposes, for data exploration purposes, and for pretty
> pictures.  We should not draw conclusions from graphs, we can only do so
> from numbers, properly controlled, properly analyzed, but numbers
> nonetheless.
>
> and,
>
> > The data may be there but if its not visibly demonstrative then your
> data is questionable when it comes to presentation.
>
> "Visibly demonstrative"?  I'm not even sure what you mean by this.  But it
> illustrates a basic mistake we all make -- we put too much emphasis on
> visualizations, on graphs.  Data is questionable based on evaluations of
> its reliability and reproducibility (statistics).  We can look at graphs to
> decide on what questions to ask of the data, but we must be cautious about
> drawing conclusions from graphs.
>
> I can show you examples of graphs which look great but are meaningless.
>  And I can show you graphs of data that look questionable but are good.
>
> Cris's basic advice was correct, but brief.  If your positives are within
> a half decade of the top end of the dynamic range, then reduce your PMT
> voltage (sensitivity).  Inserting an ND filter is not necessarily an
> equivalent (or good) solution -- it accomplishes roughly the same thing but
> at a cost of increased noise from decreased photoelectron counts.
>
> Reducing the PMT voltage won't make negatives "invisible" -- it will only
> squash them near the zero value.  Nothing is hidden.
>
> BTW, when you see your negative population go in to the "below-zero"
> measurement range of uncompensated parameters (ie towards the left edge on
> the transformed logicle scale), this is a consequence of the baseline
> restore in the DIVa electronics.  It's a pain... but it's not typically a
> problem.
>
> Just make sure that you consider ALL events below the upper end of an
> unstained sample (wherever it ends up on your settings) to be equivalent.
>  i.e., if your unstained cells go to 100, then EVERYTHING below 100,
> whether it's +10 or -300 is the same -- don't gate separately those events,
> even if they look like distinct populations in your graph.
>
> Because graphics can lie, but numbers always tell the truth.
>
> mr
>
>
>
>
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