[Cytometry] Scaling problem with FACSDiva
Dave.Dunaway at nationwidechildrens.org
Tue Feb 4 11:15:59 EST 2014
I find this to be dubious advice unless all you are interested in is crunching numbers. You show me a plot for presentation purposes where you don't "worry about the negatives" and I'll say your so called positives are meaningless. How do I know you didn't just jack voltages on a negative sample unless I can see your positives in the context of your negatives. The data may be there but if its not visibly demonstrative then your data is questionable when it comes to presentation.
From: cytometry-bounces at lists.purdue.edu [mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Cris Bare
Sent: Saturday, February 01, 2014 1:13 PM
To: Ed Podniesinski
Cc: Martina Beyrau; cytometry at lists.purdue.edu
Subject: Re: [Cytometry] Scaling problem with FACSDiva
My read on the original question is that Martina is setting a red channel
(R670) PMT voltage using the old school technique of putting a "negative"
population in the first decade.
The digital BD hardware and software is best served by adjusting PMT voltage so brightest positives are on-scale and letting the negatives fall where they may.
This is for two basic reasons: First, the untransformed (non-Biexponential) DiVa plot display by default does not display the first decade even though data certainly resides therein and is used when calculating statistics.
Second, the floating point nature of the data means that while the detector has a hard upper limit 262144 channels, the lower limit is essentially unlimited.
That means you do NOT need to set negatives arbitrarily at "300" to get valid statistics. But you do need to keep your positives below 262144 to avoid binning.
I suggest abandoning the use of unstained/isotype controls to set voltages and instead set them either based on detector efficiency (CS&T) or fully stained biological control (bright positives).
And do not worry that the "negatives" aren't visible. The data is there!
On Sat, Feb 1, 2014 at 6:34 AM, Ed Podniesinski <flow8775 at roadrunner.com>wrote:
> Have you considered trying a neutral density filter sandwiched on top
> of your R670 bandpass filter to cut down the detected photons?
> Can't remember where I got my 1" round ND filter kit from ( not at
> work right now) but I have fractional values from .5 to 3.0. Could
> have been from Thor Labs or Edmunds.
> Ed Podniesinski
> Roswell Park Cancer Institute
> -----Original Message-----
> From: cytometry-bounces at lists.purdue.edu
> [mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Martina
> Sent: Wednesday, January 29, 2014 5:36 AM
> To: cytometry at lists.purdue.edu
> Subject: [Cytometry] Scaling problem with FACSDiva
> Dear all,
> I would really appreciate some help on the following problem.
> When measuring an APC labelled antibody (to a surface protein) on
> murine neutrophils using the R670 channel on a Fortessa flow cytometer
> and FACSDiva software, both the isotype control and unstimulated
> labelled cells are set to have a median fluorescence of approx 300.
> Stimulated cells have a substantially elevated signal, and the range
> of intensities covers the whole log scale. In order to fit in the high
> intensity events the negative values are shifted to below zero, rather
> than being in line with the negative values of the isotype or
> unstimulated group. This prevents direct comparison of mean/median
> fluorescence intensities, or quantifying the % of stimulated cells
> which have a higher value than the unstimulated sample.
> Can we reset the software so that the low values are aligned, and the
> high values will be pushed up against the right hand axis if they
> exceed the available log scaling? Or increase the available log
> scaling so that the whole range of measurements will fit? I don't want
> to reduce the voltage too much as I need to keep the isotype value
> above zero.
> Thanks very much,
> Dr Martina Beyrau
> Post Doctoral Research Assistant
> Queen Mary, University of London
> E-mail: m.beyrau at qmul.ac.uk
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