[Cytometry] Scaling problem with FACSDiva

Rupak Neupane rneupane at cellerant.com
Mon Feb 10 12:03:05 EST 2014


Hi Ina,
I am not sure how different Navios is from the Gallios, but if you analyze LMD files from Gallios on Flowjo and if you uncheck that "Use FCS 2.0 information" in flowjo preference, then you can use the FCS 3.0 info from the LMD file and let Flowjo transform your data. Then, the files are no different than the ones you get from FACSDiva software.
Hope that helps.
Rupak.

-----Original Message-----
From: cytometry-bounces at lists.purdue.edu [mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Ina Laura Pieper
Sent: Thursday, February 06, 2014 9:15 AM
To: Mario Roederer
Cc: Purdue list
Subject: Re: [Cytometry] Scaling problem with FACSDiva

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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