[Cytometry] Scaling problem with FACSDiva

Cris Bare flowmail at verizon.net
Sat Feb 1 13:13:10 EST 2014


Ahhh!

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.

Why?

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!

-cbb


On Sat, Feb 1, 2014 at 6:34 AM, Ed Podniesinski <flow8775 at roadrunner.com>wrote:

> Martina,
>
> 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
>
> http://rpciflow.org
>
>
> -----Original Message-----
> From: cytometry-bounces at lists.purdue.edu
> [mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Martina Beyrau
> 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,
> Martina
>
> --
> Dr Martina Beyrau
> Post Doctoral Research Assistant
> Queen Mary, University of London
> E-mail:    m.beyrau at qmul.ac.uk
>
>
>
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