---------------------- Forwarded by David C McFarland/DEV/PHRD/SB_PLC on
07/31/2000 08:34 ---------------------------
Roederer@drmr.com on 28-Jul-2000 19:29
To: cyto-inbox
cc: David C McFarland
Subject: Re: FL3/FL4 compensation on a Calibur: Let's get it right!
I'm afraid I must disagree with Mario's disagreement. Perhaps I didn't explain
this well.
Mario said:
First I want to correct something David said which is a very common
misconception:
>Compensation is always more difficult when you have two stains that
>are vastly different in relative intensity. The more disparity, the more
>compensation needed.
This is NOT correct. Compensation is INDEPENDENT of intensity.
Compensation is ONLY related to the emission spectrum of the
fluorochromes, not how much light is being emitted. The whole
principle of compensation is based on the fact that it is independent
of intensity. Again, I would direct nonbelievers to my web pages on
compensation for a full explanation.
Suppose I'm compensating FL1 and FL2. Relatively speaking FL2 signal is 10,000
times brighter than FL1. FL1 is very dimly positive and FL2 is off scale as
compared to control. The problem arises because I have to lower the voltage on
FL2 dramatically. Especially on the Calibur, if FL2 voltage is below FL1
voltage, you have to add a LOT more compensation. I guess this means that
greater disparity in relative intensity makes compensation more difficult IF it
requires you to adjust the detector. I agree that a dim and a bright sample on
THE SAME SCALE should require the same amount of compensation. I've read all
about that. (And I also agree that everyone should take a look at Marios site
on compensation.)
Mario also said:
Furthermore, your remaining explanation requires correction and comments:
>Your FL4-%FL3 problem is exacerbated by the fact that it
>isn't JUST a compensation problem. Compensation is used to remove the
>fluorescence contribution due to spectral overlap among fluors. For
instance,
>you have to compensate your "green"(FITC/FL1) dye from the "orange"
>(PE/FL2)channel because it has an orange "tail". We express this as
>FL2-30%FL1,
>for example. What we're saying is that an amount of light equal to 30% of
the
>light detected in FL1 is showing up in FL2 and we need to ignore that much
of
>the contribution to signal in that detector. The problem you're seeing
isn't
>due to to spectral overlap. You see, the CY5 portion of your tandem dye,
>tricolor, is excited very well by your little red diode laser. It's
"real"
>fluorescence in FL4. (Imagine trying to compensate PE fluorescence
>out of FL2.
>It doesn't work so well.) So, the reason your FL4(APC?) signal is being
>ablated is because it is lower than the siganl you are getting from
tricolor.
>If you compensate the tricolor signal in FL4, you are knocking your dim
FL4
>signal off scale. You are also correct that there is a time delay
calculation
>that has to be performed by the cytometer to ensure that signal collected
when
>cells strike the first laser is correlated with signal from excitation by
the
>second. You should run FACSComp and do the time delay calibration every
time
>you are using the diode laser. If you're not using FL4, turn it off.
This is also mostly incorrect. There is no difference between "real"
and any other kind (including "tails") of fluorescence. The
fluorescence of Cy5PE in the FL2 channel are from photons emitted
from the PE molecule. The fluorescence in the FL3 channel are some
of the very "red" photons emitted from the PE molecule, but primarily
those which come from the Cy5 through resonance energy transfer from
the PE molecule. Finally, the fluorescence in FL4, as you correctly
note, is from photons coming from direct excitation of the Cy5
molecule by the second laser.
My retort: OK. once again I agree, that according to the detector there is no
difference between a photon coming from your dye of choice, autofluorescence,
whatever. I was trying to explain it in a way that a non-expert could grasp.
When I said "real", I meant that what he is having a hard time compensating is
not the normal spectral overlap like the orange signal you get from fluorescein.
The signal seen in FL4 is due the excitation of CY5 by the red laser and SHOULD
be there.
I made the mistake of saying: (Imagine trying to compensate PE fluorescence
out of FL2.
It doesn't work so well.) Perhaps I should have said "fluorescein out of FL1"?
Once again, I realize that from your point of view PE IS getting compensated out
of FL2, being a component of tricolor. So, you put on your tricolor single
positive compensation control and get lots of orange signal in FL2 and
compensate it out. If I were explaining to someone how to do compensation, I
would tell them that they are compensating tricolor, not PE. Mentioning tandem
conjugates and all that just seems to muddy the water. Sine the original
inquisitor didn't have a problem with compensation in that direction, I ignored
it. So, what I really meant when I said "Imagine trying to compensate PE
fluorescence out of FL2" was "imagine running your PE single positive
compensation control and trying to compensate the signal out of FL2". This is
basically what you are doing if you are trying to compensate FL4 while running
tricolor.
Mario continues: It is necessary to realize that "spectral overlap" refers
not only to
emission overlap, but to excitation overlap as well.
Fluorescence is
composed of two distinct spectra: excitation and emission!
I'll add my own Amen here.
Finally, the time delay calculation is irrelevant in this whole
process.... As long as it was correctly set at the time of
collecting the compensation controls (or bead calibration), then
it
will NOT affect the ability to properly compensate the samples.
Notice the big IF in the above statement. Time delay calibration is irrelevant
IF it was correctly performed.
Boy, this is much easier when you are sitting in front of an instrument and
SHOWING the person how to do it! Anybody else want to muddy the waters?
And that's all I have to say about that?
David McFarland
SmithKline Beecham Pharmaceuticals
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