Calculated ratios & more questions

Ray Hicks rh208 at cus.cam.ac.uk
Fri Feb 6 08:17:02 EST 1998


Darren,

FCS Assistant assumes (because I do) that you'll be acquiring and ratioing
linear data.  The reasons for this are:

1) Log data, while it allows you to squash a larger range of values into
the same number of "channels", does so at the expense of precision.  The
first 256 channels cover the range 1 to 10 with probably more precision
than is needed given the amount of noise in this region, while the last 256
channels cover the range 1000 to 10000, which means that there are large
gaps between the discrete values that can be recorded in this region.

2) Log amplifiers aren't perfect, and you don't necessarily reconstruct the
input by "linearising" the output.  They're best for increasing the dynamic
range for enumerative work such as immunophenotyping, where intensity is
used qualitatively.

3) The heterogeneity of cell loading isn't very wide with any of the AM
coupled calcium dyes, nor is the change in intensity on calcium binding.
It is easily possible to fit the range of intensities in any population
I've come across onto a linear scale.

I might include a log ratio option in a future version (as you spotted, the
result of dividing two log values isn't what's needed), but I recommend
that you try varying your loading regime to enable you to acquire in
linear, you'll get better results.

If you have any other concerns about FCS Assistant, I'd appreciate it if
you get in touch with me in the first place.

Here's a quick "how-to" for using your FACStar plus:

The pulse processing boards on FACStar plus and Vantage machines come
between the preamplifier, allowing fine gain control to be used, but come
before the log or programmable linear amplifiers.  The signals that they
receive are those that you see when you use a gain of one.  If you use log
or higher than unity linear gain to make your signals look better on
screen, it has no effect on the pulse processor input, and the resulting
ratio will look the same.  The pulse processors don't work too well with
low level signals, so any event with a signal in channel 50 or less is
forced to maximum ratio.

Setting it so it "looks good" does work, but only if you've got linear gain
set to one, and use the fine gain control and PMT voltage to change the
signal levels:

1) Set up a dot plot with violet versus blue/green fluorescence. With
resting loaded cells set the PMT of your blue/green detector so that the
main population covers most of the blue/green axis.  You'll probably have
some cells that have much less loading than the main population, these are
erythrocytes and leaky dead cells mainly, make sure that these don't occupy
too much of the axis.

2) With the same cells, set the violet detector's gain so that the main
population  occupies the first quarter or so of the violet axis, make sure
that the signals from loaded cells are greater than channel fifty. Keeping
the violet population in the lower part of the scale leaves room for the
increase on calcium binding.

3) draw a gate that ignores all events that lie around the edges of the
trace, as well as those in the bottom left corner, this gets rid of
artefactual maximum-ratio events

4) Set up a histogram of ratio (using the above gate) and vary its gain to
put its mean somewhere around channel 100. You can manipulate the PMTs to
fine tune its position, but make sure that you don't drop them so low or
raise them so high that the signals go out of the gate

5) Add some ionomycin to your cells to give a large response, check that
the violet fluorescence doesn't go off-scale, if it does then drop the PMT
volts.  Check that the ratio doesn't go off scale, if it does then drop its
gain.

Let me know if you'd like some pictures to clarify the above, and I'll put
them on my web page,

Ray


At 17:26 -0500 4/2/98, Darren Hickerson wrote:
>Kirk & others:
>I am using calculated ratios on a FACScan using a visible laser Ca
>technique analogous to Indo-1.  I have compared the results to those
>obtained on FCS Assistant.  The biggest problem I see with FCS Assistant
>is that it seems to use the superimposed "1024" channel axis that the data
>is (are) actually saved in to calculate the ratios, rather than the 10,000
>channel true fluorescence scale that reflcets actual intensity values, so
>the final ratios are not "real" values.  The pictures are very nice, but
>the numbers don't reflect the real changes.  Hand-calculating the
>intensities seems to be the best way (true FL"A" divided by true FL"B" for
>a given time point -- you just lose the flow of the kinetic scale this
>way).  Phoenix Flow has some software that SEEMS like it would do this for
>you (from the pix on the ads), but I'm not sure.
>
>Anyone else with advise or how to get "real" ratios on FCS Assistant, or
>with more detailed info on Phoenix software, please write in.
>
>Also, if anyone has a quick "how-to" on setting up the real ratio
>parameters on a FACStar Plus, I'm all ears.  The manual gets so bogged
>down in calibrating the axis, I'm not sure if just setting it so it "looks
>good" even works.  Same questions again: can I use "log" scale FL
>parameters, or do they all have to be set to the 1024 scale before
>acquisition?  I asked this before & never got clear on what people were
>calling linear, log, channel, and "true fluorescence" values -- everyone
>seems to use different terminology.
>
>Thanks, Darren Hickerson
>
>dhickerson at brody.med.ecu.edu
>
>Core Flow Cytometry Facility, Brody 4W37
>Department of Microbiology and Immunology
>East Carolina University School of Medicine
>Greenville, NC 27858
>
>Phone: (919) 816-2799
>Fax: (919) 816-5018


                              Ray Hicks
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