[RASMB] Photomultiplier tube disturbances?

[Borries Demeler]

> 
> Troy, have you read the past RASMB discussions of problems arising from
> trying to run samples in the reference channel? Remember that the instrument
> is designed to scale the photomultiplier voltage and the programmable gain
> amplifier based on the intensity it sees at 6.5 cm before it starts each
> radial scan. 

John,

from what I see it is not entirely clear that this is the problem in
this case.  UltraScan will give you a plot of the intensity over time by
averaging the intensity for water scans. I have seen two cases now where
the culprit was not absorbance in the reference channel, but clearly
a problem with the intensity fluctuating wildly. A replacement data
acquisition board fixed the problem in one case, a new monochromator in
another. There could be other causes.

> It seems to me you are seeing exactly what has been discussed previously:
> when the DNA boundary in the reference channel sediments to near 6.5 cm, the
> instrument is readjusting the PMT voltage and/or the PGA gain, and this
> produces a break in the pseudo-absorbance data for both channels of that
> cell. Basically pseudo-absorbance only works correctly if the instrument
> ends up not changing the PMT voltage or PGA setting for that cell at all
> during the run; if the instrument decides it needs to do so, either because
> something is sedimenting in the reference, or just because the lamp
> intensity is drifting for some reason, you are screwed.

...lamp intensity should of course not drift so much that it affects the 
quality of the data. In absorbance mode you are only caching the problem
as stochastic noise increases with decreased intensity. This may be very hard
to pick up, but looking at the intensity over time will reveal if some hardware
problem exists, or if your buffer absorbs, adding to the absorbance of your sample.

> Whenever you run in pseudo-absorbance mode you are asking the instrument to
> do something it wasn't ever intended to do. When the OD in the reference
> channel is quite low it usually works okay, but you are risking getting low
> quality or un-interpretable data. You also put yourself in a position where
> you can't complain to Beckman that something is wrong.

I must say that we have gotten consistently *excellent* results with this approach
and I haven't run a velocity run in absorbance mode in years. The biggest
benefit from doing what Troy does is a reduction of stochastic noise by a 
factor of 1.4, but also running a low concentration sample in the reference
channel is nice since you don't have to pay the price for the additional
scan time and increase the capacity of the machine. So, yes, there is a risk
that you screw up your data if your absorbance is too high, but that is 
easily enough controlled, and the benefits far outweigh the downside.
I'll attach an image for a side-by-side comparison of absorbance vs. intensity
of the same dataset. Note the total OD and the difference in stochastic noise.
 
> I don't know if Tom Laue's AOS operating software can get into the machine
> internals and modify this re-scaling behavior. I tend to doubt it, but if it
> could that would help tremendously when running in pseudo-absorbance mode.

As far as I understand it the AOS currently only works with the FDS from Aviv, not
with the Beckman absorbance system.

> As Borries said, probably the only way to get around this issue is to lower
> the OD in the reference channel, or just run in normal absorbance mode with
> buffer in the reference.

...or in intensity mode with water in the reference (provided your machine
works properly, if not, you should get it fixed anyway).

-b.
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