[Cytometry] Scaling problem with FACSDiva
roederer at drmr.com
Wed Feb 5 21:28:44 EST 2014
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.
> 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.
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